Am J Physiol Heart Circ Physiol
279: H2829–H2837, 2000.
Mechanisms by which bradykinin promotes fibrosis in
vascular smooth muscle cells: role of TGF-␤ and MAPK
CHRISTELLE D. DOUILLET,1 VICTORIA VELARDE,1 JULIE T. CHRISTOPHER,1
RONALD K. MAYFIELD,1,3 MARIA E. TROJANOWSKA,1 AND AYAD A. JAFFA1,2
Departments of 1Medicine and 2Cell and Molecular Pharmacology and Experimental Therapeutics,
Medical University of South Carolina, and 3Ralph H. Johnson Veteran Affairs Medical Center,
Charleston, South Carolina 29425
Received 17 February 2000; accepted in final form 25 July 2000
(VSMC) expansion and
the resultant increased extracellular matrix (ECM)
deposition play pivotal roles in the progression of
atherogenesis (2). Increases in ECM components such
as collagens types I and IV are found in plaque areas of
aortas from type 1 diabetic patients (32). These
changes in matrix composition may impair vascular
function by changing VSMC from their contractile
state to a synthetic state (5). Although the precise
mechanisms underlying these changes are undefined,
the interaction between arterial smooth muscle cells
and the surrounding matrix suggests a dynamic system in which changes in composition and spatial organization of matrix itself may profoundly change the
metabolic and proliferative activity of VSMC (34, 42).
Collagen formation is the major contributor to the
growth of atherosclerotic lesions (2). Of the various
types of collagens, type I is the primary component of
atherosclerotic plaque and is synthesized by arterial
smooth muscle cells in response to growth factors (2).
Thus matrix deposition is a characteristic part of the
sclerotic process that contributes to vascular function
impairment by disrupting normal cell-cell interaction
and modifying tissue elasticity.
Among the factors that play a central role in vascular fibrosis is transforming growth factor-␤ (TGF-␤).
TGF-␤ has been shown to stimulate collagen expression in VSMC and also to influence the expression of
some matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs). Since matrix deposition is the
result of a balance between synthesis and degradation
of ECM components, it is conceivable that the delicate
balance between MMP and TIMP, as well as the rate of
ECM synthesis, determine the final matrix accumulation.
The localization of components of the kallikreinkinin system within the vascular wall suggests this
system has a role in the regulation of vascular tone,
hypertension, and atherogenesis (22, 24). Kallikrein
and its mRNA are expressed in isolated arteries and
veins and in cultured VSMC (29, 33). Kininogen, the
substrate for kinin generation by kallikrein, kininase
activity, and B2-kinin receptors are present in VSMC
(30). Thus locally generated kinins can act in an autocrine or paracrine fashion to influence vascular function. The physiological actions of bradykinin (BK) are
mediated via generation of second messengers such as
nitric oxide and eicosanoids (17, 36). BK relaxes VSMC
Address for reprint requests and other correspondence: A. A. Jaffa,
Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical Univ. of South Carolina, 114 Doughty St., PO Box 250776,
Charleston, SC 29425 (E-mail: [email protected]).
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.
collagen; tissue inhibitor of metalloproteinase 1; transforming growth factor-␤; mitogen-activated protein kinase
VASCULAR SMOOTH MUSCLE CELL
http://www.ajpheart.org
0363-6135/⫺1900 $5.00 Copyright © 2000 the American Physiological Society
H2829
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Douillet, Christelle D., Victoria Velarde, Julie T.
Christopher, Ronald K. Mayfield, Maria E. Trojanowska, and Ayad A. Jaffa. Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells:
role of TGF-␤ and MAPK. Am J Physiol Heart Circ Physiol
279: H2829–H2837, 2000.—Accumulation of extracellular
matrix (ECM) is a hallmark feature of vascular disease. We
have previously shown that hyperglycemia induces the expression of B2-kinin receptors in vascular smooth muscle
cells (VSMC) and that bradykinin (BK) and hyperglycemia
synergize to stimulate ECM production. The present study
examined the cellular mechanisms through which BK contributes to VSMC fibrosis. VSMC treated with BK (10⫺8 M)
for 24 h significantly increased ␣2(I) collagen mRNA levels.
In addition, BK produced a two- to threefold increase in ␣2(I)
collagen promoter activity in VSMC transfected with a plasmid containing the ␣2(I) collagen promoter. Furthermore,
treatment of VSMC with BK for 24 h produced a two- to
threefold increase in the secretion rate of tissue inhibitor of
metalloproteinase 1 (TIMP-1). The increase in ␣2(I) collagen
mRNA levels and ␣2(I) collagen promoter activity, as well as
TIMP-1 secretion, in response to BK were blocked by antitransforming growth factor-␤ (anti-TGF-␤) neutralizing antibodies. BK (10⫺8 M) increased the endogenous production
of TGF-␤1 mRNA and protein levels. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059
inhibited the increase of ␣2(I) collagen promoter activity,
TIMP-1 production, and TGF-␤1 protein levels observed in
response to BK. These findings provide the first evidence that
BK induces collagen type I and TIMP-1 production via autocrine activation of TGF-␤1 and implicate MAPK pathway as
a key player in VSMC fibrosis in response of BK.
H2830
REGULATION OF VSMC FIBROSIS BY BK
METHODS
Cell culture. Rat aortic VSMC from male Sprague-Dawley
rats (Charles River Laboratories, Wilmington, MA) were
prepared by a modification of the method of Majack and
Clowes (21). A 2-cm segment of thoracic aorta cleaned of fat
and adventitia was incubated in 1 mg/ml collagenase for 3 h
at room temperature. The aorta was then cut into small
sections and placed into a culture flask for explantation in
minimal essential media (MEM) containing 10% FBS, 1%
nonessential amino acids, 100 mU/ml penicillin, and 100
␮g/ml streptomycin (Cellgro; Mediatech, Herndon, VA). Cells
were incubated at 37°C in a humidified atmosphere of 95%
air-5% CO2. Medium was changed every 3–4 days, and cells
were passaged every 6–8 days by harvesting with trypsinEDTA. Cell viability was assessed by standard dye exclusion
techniques using 1% trypan blue. VSMC were identified by
the following criteria. They stained positive for intracellular
cytoskeletal fibrils of ␣-actin and smooth muscle cell-specific
myosin (indicative of contractile cell) and negative for factor
VIII antigens. VSMC isolated by this procedure were homogenous and were used in all studies between passages 2 and 6.
RNA extraction and Northern blotting. VSMC in 15-cm
dishes were rendered quiescent by growing them in serumfree media for 48 h. Cells were then stimulated with agonists
as indicated. Total RNA from the cells was extracted by the
chloroform-phenol method (6). RNA concentration and purity
were determined spectrophotometrically (Ultrospec III,
Pharmacia) by absorbance at 260 and 280 nm. Total RNA (20
␮g) was denatured at 65°C for 15 min and ran on 1.5%
agarose gel in denaturing conditions. The gel was stained
with ethidium bromide to determine the position of the 28S
and 18S ribosomal RNA and to demonstrate that similar
amounts of intact RNA were used for each sample. Total
RNA was transferred from the gel to Nytran membrane
filters (Schleicher and Schuell, Keene, NH) by a pressure
transducer (PosiBlot; Stratagene, La Jolla, CA), prehybridized for 2 h, and hybridized at 55°C for 18–24 h with nicktranslated cDNA probes labeled with 32P using a nick-translation kit (Bethesda Research Laboratories, Bethesda, MD).
The hybridized membranes were washed and exposed to film.
Autoradiographs (Kodak XAR-5 film, Eastman Kodak, Rochester, NY) of the membranes were obtained and scanned, and
the intensities of the bands were quantified by NIH image
1.61/68k.
TGF-␤1 protein level determination. VSMC were cultured
in six-well plates (9.6 cm2/well). At 70% confluence, cells were
serum starved by the changing of the serum-free media four
times within 24 h. Cells were then stimulated for 48 h with
10⫺8 M BK, in presence or absence of 40 ␮M of PD-98059,
in exactly 1.5 ml. TGF-␤1 protein levels were determined
by colorimetric enzyme-linked immunosorbent assay kit
(ELISA; R&D Systems, Minneapolis, MN) in the conditioned
media that were activated by HCl (to measure both active
and latent TGF-␤1) according to the manufacturer instructions, and expressed as picograms per milliliter.
Transfection of VSMC with the ␣2(I) collagen promoter.
VSMC were transfected by the calcium chloride method with
a plasmid containing the ␣2(I) collagen promoter attached to
the chloramphenicol acetyl transferase (CAT) gene (⫺353
COL1A2 CAT); the plasmid was obtained from Promega
(Madison, WI), prepared as described by Tamaki et al. (41),
purified by double cesium chloride purification, and stored at
4°C. The plasmid (20 ␮g of plasmid DNA) in a 2 M CaCl2
solution was mixed with an equal volume of 2⫻ DNA precipitation buffer (3 Prime 35 Prime Inc., Boulder, CO) and
incubated for 30 min before addition to the cells. After 16 h of
incubation, the culture medium was changed to 1% FBS in
MEM. Two hours later, the cells were stimulated for 24 h as
indicated. The cells were then washed in PBS and resuspended in 0.25 M Tris, pH 8.0. The CAT was extracted from
the cells by four cycles of freezing-thawing and centrifugation
at 13,800 g for 15 min. Protein concentrations were determined in each cell extract by Bio-Rad protein assay (Bio-Rad,
Hercules, CA), and 10–20 ␮g of proteins from each dish were
used for CAT activity assay. CAT activity of each sample was
performed in presence of 2.5 mg/ml of butyryl-CoA and
[14C]chloramphenicol incubated for 90 min at 37°C. Acetylated chloramphenicol was extracted by tetramethylpentadecane/xylene (2:1), and radioactivity was measured in a BetaMax counter (ICN, Costa Mesa, CA). In some experiments, 5
␮g of the pSV-␤-galactosidase control vector (Promega) was
cotransfected to normalize for transfection efficiency. ␤-Galactosidase activity assays were measured according to the
manufacturer instructions
Western blotting of TIMP-1. VSMC in 60-mm dishes were
rendered quiescent by growing them in serum-free media for
24 h. Cells were then stimulated as indicated. The conditioned media were collected after 24 h and concentrated by
lyophilization. Proteins were determined by Bio-Rad protein
assay and resolved on SDS-PAGE. The separated proteins in
the gel were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and immunoblotted with goat polyclonal
anti-rat TIMP-1 specific antibody (1:2,000 dilution; Santa
Cruz Biotechnology, Santa Cruz, CA). The blots were revealed by anti-goat IgG antibody conjugated with alkaline
phosphatase (1:6,000 dilution, Santa Cruz Biotechnology)
and CDP-Star chemiluminescent system (NEN Life Sciences
Products, Boston, MA). The membranes were exposed to film
(X-Omat LS, Kodak). Films were scanned, and the intensities
of the bands were quantified by NIH image 1.61/68k.
Phospho-MAPK immunoblots. To measure MAPK activity,
20–25 ␮g of soluble protein obtained from cell lysates were
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through synthesis and release of nitric oxide from the
endothelium, but in states of vascular injury in which
endothelial integrity is compromised, BK can act directly on VSMC, to increase intracellular calcium and
cause contraction (4).
The signaling pathways leading to VSMC fibrosis
are just beginning to be studied and seem to be derived
from multiple sources. Our recent data demonstrate
that BK stimulates early mitogenic signals in VSMC.
Through activation of its B2 receptors, BK stimulates
activation and nuclear translocation of p42mapk and
p44mapk and induces expression of c-fos and c-jun
mRNA levels and formation of the AP-1 complex (11,
44). The cellular signals through which BK stimulates
p42mapk and p44mapk activation and c-fos mRNA expression in VSMC involves activation of a calcium/
calmodulin pathway, src kinase, protein kinase C, and
generation of reactive oxygen species (14, 28, 44).
Therefore, the present studies were designed to explore the role of BK in mediating VSMC fibrosis. We
found that BK induces ␣2 chain of type I [␣2(I)] collagen
mRNA levels, ␣2(I) collagen promoter activity, and
TIMP-1 production via autocrine activation of TGF-␤1.
In addition, our findings demonstrate that BK signals
via the mitogen-activated protein kinase (MAPK) pathway to mediate its effects on ␣2(I) collagen and TIMP-1
production. These findings provide the first evidence
for a potential role for BK in VSMC fibrosis.
REGULATION OF VSMC FIBROSIS BY BK
RESULTS
Induction of ␣2(I) collagen expression by BK. To investigate whether BK induces ECM formation, we
measured ␣2(I) collagen mRNA expression in VSMC
treated with BK (10⫺8 M) for 24 h. As shown in Fig. 1A,
BK produced a significant increase in ␣2(I) collagen
mRNA levels, expressed relative to glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA. The ␣2(I)
collagen mRNA levels were increased to 160% of control VSMC (BK vs. control, P ⬍ 0.003, n ⫽ 3).
We next assessed whether BK can induce transcriptional activation of ␣2(I) collagen gene by measuring its
effect on the activity of ␣2(I) collagen promoter. VSMC
transfected with a plasmid containing the ␣2(I) collagen promoter were stimulated for 24 h with either BK
(10⫺8 M) or TGF-␤1 (1 ng/ml) as positive control. The
CAT activity (used as a reporter for the activity of the
collagen promoter) was determined in the cell lysate.
The results (Fig. 1B) demonstrate for the first time
that BK can directly increase the activity of the colla-
gen promoter. BK produced a 2.5-fold increase in ␣2(I)
collagen promoter activity, whereas TGF-␤1 produced
a fourfold increase (control vs. BK or TGF-␤1, P ⬍
0.005, n ⫽ 8 experiments).
Induction of TGF-␤1 expression by BK. To define the
cellular mechanisms through which BK increased collagen expression, the ability of BK to stimulate the
endogenous production of TGF-␤ was examined. We
measured the TGF-␤1 mRNA levels in VSMC treated
with BK (10⫺8 M) for various times. BK treatment
resulted in a significant increase in TGF-␤1 mRNA
levels/GAPDH mRNA levels above control. This increase in TGF-␤1 was observed as early as 4 h, maintained at 6 h, and returned to normal values by 24 h
(P ⬍ 0.02, BK vs. 0 h, n ⫽ 4 experiments, Fig. 2).
Role of TGF-␤ in BK-induced ␣2(I) collagen expression. To further define the role of TGF-␤ in BK signaling, we examined whether the increase in ␣2(I) collagen mRNA levels or promoter activity in response to
BK are mediated via autocrine activation of TGF-␤.
VSMC were treated with BK (10⫺8 M) for 24 h in the
presence and absence of neutralizing anti-TGF-␤ antibodies (20 ␮g/ml, R&D Systems). This concentration of
anti-TGF-␤ antibody has been shown to neutralize the
effects of TGF-␤ on mesangial cell proliferation and
matrix formation (39, 45). The results shown in Fig. 3A
indicate that the ability of BK to induce ␣2(I) collagen
mRNA levels is blocked by the anti-TGF-␤ neutralizing
antibody (P ⬍ 0.05, BK vs. BK⫹TGF-␤ neutralizing
antibody, n ⫽ 3). We also examined the effects of
TGF-␤ neutralizing antibody on VSMC transfected
with the ␣2(I) collagen promoter and stimulated with
BK (10⫺8 M) for 24 h. Although we have observed
differences in the magnitude of response to BK between different cell lines, once again, BK produced a
significant increase in CAT activity compared with
unstimulated control cells (BK vs. control, P ⬍ 0.01,
n ⫽ 5, Fig. 3B). This increase in CAT activity in
Fig. 1. Bradykinin (BK) stimulates the expression of
␣2(I) collagen in vascular smooth muscle cells (VSMC).
A: quiescent VSMC were stimulated with BK (10⫺8 M)
for 24 h. RNA was extracted from cells, and ␣2(I) collagen and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) levels were measured by Northern blot analysis. The bar graph represents the relative intensities
of ␣2(I) collagen to GAPDH mRNA levels. Blot is representative of 3 separate experiments. B: VSMC were
transfected with a plasmid containing ␣2(I) collagen
promoter and chloramphenicol acetyl transferase
(CAT) gene as a reporter for the activity of the ␣2(I)
collagen. Transfected cells were stimulated for 24 h
with BK (10⫺8 M) or transforming growth factor-␤1
(TGF-␤1, 1 ng/ml) as a positive control. Bar graph
represents the ␣2(I) collagen promoter CAT activity
expressed as a percentage of control. Results are representative of 8 separate experiments. *P ⬍ 0.005 vs.
control.
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subjected to SDS-PAGE. The separated proteins in the gel
were transferred to polyvinylidene difluoride membranes,
and immunoblotted with rabbit polyclonal phospho-specific
MAPK antibodies that specifically recognize tyrosine phosphorylated p42mapk and p44mapk (New England Biolabs, Beverly, MA). The phospho-MAPK antibody was used at 1:6,000
dilution, whereas the control antibody, which recognizes total MAPK, was used at 1:4,000 dilution. The membranes
were incubated overnight with the antibody buffer (TBS,
0.05% Tween 20, 1% BSA), washed in TBS-0.05% Tween 20,
and exposed to goat anti-rabbit horseradish peroxidaseconjugated IgG (1:5,000) in antibody buffer for 1 h. Immunoreactive bands were visualized by a chemiluminescent method
(Renaissance, New England Biolabs) using Kodak X-LS
film.
Statistical analysis. Data are expressed as means ⫾ SE
and were analyzed by ANOVA and by using student’s t-test
for unpaired analysis. Differences were considered significant if P ⬍ 0.05.
H2831
H2832
REGULATION OF VSMC FIBROSIS BY BK
response to BK stimulation was completely blocked in
the presence of anti-TGF-␤ neutralizing antibody. Anti-TGF-␤ neutralizing antibody had no significant effect on basal CAT activity (Fig. 3B).
BK increases TIMP-1 secretion via a TGF-␤-dependent mechanism. TIMPs are proteins that regulate the
activity of MMPs and play an integral part in the
turnover rate of ECM under physiological and pathological conditions (13). Thus it is conceivable that
VSMC matrix accumulation is the net result of signals
that promote increased TIMP activity. To address this
possibility, we examined the effects of BK on the secretion rate of TIMP-1 in VSMC and explored the role of
TGF-␤ in mediating the effects of BK on TIMP-1 production. VSMC were stimulated for 24 h with either
BK (10⫺8 M) or TGF-␤1 (1 ng/ml), as positive control, in
the presence and absence of anti-TGF-␤ neutralizing
antibody (20 ␮g/ml). The conditioned media were analyzed by Western blotting for TIMP-1 using specific
anti-TIMP-1 antibody (1:2,000 dilution, Santa Cruz
Biotechnology). The results shown in Fig. 4 demonstrate that both BK and TGF-␤1 produced a significant
increase in TIMP-1 production in VSMC (P ⬍ 0.05,
control vs. BK or TGF-␤1, n ⫽ 5). This increase in
TIMP-1 production in response to either BK or TGF-␤1
was completely blocked by the anti-TGF-␤ neutralizing
Fig. 3. BK induces ␣2(I) collagen expression
via autocrine activation of TGF-␤. A: quiescent VSMC were stimulated for 24 h with
10⫺8 M BK in presence or absence of 20 ␮g/ml
of neutralizing anti-TGF-␤ antibody (TGF-␤
NA). RNA was extracted from cells and ␣2(I)
collagen and GAPDH mRNA levels were measured by Northern blot analysis. Values represent relative intensities of ␣2(I) collagen
mRNA to GAPDH mRNA levels. B: VSMC
were transfected with a plasmid containing
␣2(I) collagen promoter and CAT gene as a
reporter for the activity of the ␣2(I) collagen.
Transfected cells were stimulated for 24 h
with 10⫺8 M BK in presence or absence of 20
␮g/ml of neutralizing anti-TGF-␤ antibody
(TGF-␤ NA). Bar graph represents the ␣2(I)
collagen promoter CAT activity expressed as
percentage of control and is representative of
5 separate experiments. *P ⬍ 0.01 vs. control.
†P ⬍ 0.05 vs. BK.
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Fig. 2. Induction of TGF-␤ mRNA levels by BK in VSMC. Quiescent
VSMC were stimulated with BK (10⫺8 M) for various times (0, 4, 6,
8, and 24 h). RNA was extracted from cells, and TGF-␤1 and GAPDH
mRNA levels were measured by Northern blot analysis. Top: relative
intensities of TGF-␤1 to GAPDH mRNA levels. Bottom: blot representative of 4 separate experiments. *P ⬍ 0.02 vs. 0 h (control).
Fig. 4. BK stimulates tissue inhibitor of metalloproteinase 1
(TIMP-1) secretion via autocrine activation of TGF-␤. VSMC were
stimulated with 10⫺8 M BK or 1 ng/ml of TGF-␤1 in presence or
absence of 20 ␮g/ml of neutralizing anti-TGF-␤ antibody (TGF-␤
NA). Conditioned medium was collected after 24 h, and equal
amounts of proteins were resolved on SDS-PAGE and immunoblotted with a polyclonal antibody against rat TIMP-1. Top: intensities of
TIMP-1 bands expressed as percentage of control. Bottom: representative blot of 5 separate experiments. *P ⬍ 0.05 vs. control. †P ⬍ 0.02
vs. BK. #P ⬍ 0.05 vs. TGF-␤.
REGULATION OF VSMC FIBROSIS BY BK
antibody, whereas the antibody alone had no effect on
basal TIMP-1 production (Fig. 4).
Activation of p42mapk and p44mapk by BK. MAPKs
belong to the group of serine-threonine kinases that
are rapidly activated in response to growth factors and
appear to integrate multiple intracellular signals
transmitted by various second messengers. To understand the signal transduction events mediated by BK,
we examined its effects on MAPK activation. Treatment of VSMC with 10⫺8 M BK for 5 min resulted in a
rapid increase in tyrosine phosphorylation of p42mapk
and p44mapk, compared with unstimulated cells (Fig.
5). However, in the presence of the MAPK kinase
inhibitor PD-98059 (40 ␮M), the increase in MAPK
phosphorylation in response to BK was reduced. This
finding is consistent with our previous observations
demonstrating that BK activates MAPK in a concentration and time-dependent manner (28, 44).
Induction of TGF-␤1 by BK is mediated via a MAPKdependent pathway. To determine whether the increase in TGF-␤1 mRNA in response to BK translates
into an increase in TGF-␤1 protein levels, TGF-␤1
protein levels were measured in the conditioned media
of VSMC stimulated with BK (10⫺8 M). As shown in
Fig. 6, BK treatment resulted in a significant increase
in TGF-␤1 levels, compared with unstimulated control
cells (84.5 ⫾ 6.1 vs. 30.8 ⫾ 2.9 pg/ml, BK vs. control,
respectively, P ⬍ 0.0001, n ⫽ 5). To evaluate whether
activation of MAPK modulates the BK-induced increase in TGF-␤1 protein levels we observed, VSMC
were treated with BK (10⫺8 M) in the presence of the
MAPK inhibitor, PD-98059. The results shown in Fig.
6 demonstrate that inhibition of the MAPK pathway by
PD-98059 completely eliminates the response of BK to
stimulate TGF-␤1 protein levels (84.5 ⫾ 6.1 vs. 37.7 ⫾
2.7 pg/ml, BK vs. BK⫹PD-98059, P ⬍ 0.0001, n ⫽ 5).
Role of MAPK in BK-induced ␣2(I) collagen and
TIMP-1 expression. To examine whether activation of
p42mapk and p44mapk modulates the increase in ␣2(I)
collagen promoter activity in response to BK, VSMC
transfected with the ␣2(I) collagen promoter were pretreated with the MAPK kinase inhibitor PD-98059 (40
␮M) for 30 min, followed by BK (10⫺8 M) stimulation
for 24 h. In the absence of PD-98059, BK produced a
significant increase in CAT activity compared with
unstimulated control cells (BK vs. control, P ⬍ 0.001,
n ⫽ 5, Fig. 7A). This increase in CAT activity in
response to BK stimulation was reduced significantly
in the presence of MAPK kinase inhibitor (P ⬍ 0.04,
BK vs. BK⫹PD-98059, Fig. 7A). The MAPK kinase
inhibitor had no significant effect on basal CAT activity
(Fig. 7A).
To examine whether p42mapk and p44mapk also modulate the increase in TIMP-1 secretion in response to
BK, VSMC were pretreated with the MAPK kinase
inhibitor PD-98059 (40 ␮M) for 30 min, followed by BK
(10⫺8 M) stimulation for 24 h. The release of TIMP-1
protein levels into the conditioned media was measured by Western blots using specific anti-TIMP-1
antibodies (1:2,000 dilution). BK produced a threefold
increase in the secretion rate of TIMP-1 into the media
compared with unstimulated cells (BK vs. C, P ⬍ 0.05,
n ⫽ 4) (Fig. 7B). However, inhibition of MAPK activity
significantly decreased TIMP-1 secretion in response to
BK (BK vs. BK⫹PD-98059, P ⬍ 0.05, n ⫽ 4). The
Fig. 6. Induction of TGF-␤1 protein secretion by BK in VSMC.
Quiescent VSMC were stimulated with BK (10⫺8 M) for 48 h in
presence or absence of MEK inhibitor PD-98059 (40 ␮M). Conditioned media were collected and activated to determine the total
level of TGF-␤1 protein, using a colorimetric ELISA kit assay. Values
are means ⫾ SE of TGF-␤1 protein levels (expressed in pg/ml) and
are representative of 5 separate experiments. *P ⬍ 0.0001 vs. control. #P ⬍ 0.0001 vs. BK.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on June 17, 2017
Fig. 5. Phosphorylation of p42mapk and p44mapk by BK in VSMC.
VSMC were stimulated with BK (10⫺8 M) for 5 min in the presence
and absence of MEK inhibitor PD-98059 (40 ␮M). Mitogen-activated
protein kinase (MAPK) phosphorylations (p42mapk and p44mapk)
were measured by immunoblot using anti-phosphotyrosine-MAPK
antibodies (P-MAPK), and total MAPK was measured in the same
immunoblot by stripping the membrane and immunoblotting with
anti-total MAPK antibodies (T-MAPK). Bottom: blots are representative of 6 separate experiments. Top: intensities of both p42mapk and
p44mapk bands measured in a densitometer relative to total MAPK
and expressed as percent phosphorylation relative to control. *P ⬍
0.01 vs. control. †P ⬍ 0.05 vs. BK.
H2833
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REGULATION OF VSMC FIBROSIS BY BK
MAPK kinase inhibitor did not significantly alter basal
secretion rate of TIMP-1.
DISCUSSION
The role of locally generated vasoactive factors in
regulating vascular tone and structure under normal
and pathological conditions has been increasingly emphasized. Among these factors, BK and angiotensin II
are often considered to have opposite effects on vessel
wall with regard to vascular reactivity and arterial
compliance. Although the role of angiotensin II has
been extensively studied with regard to vascular injury
and remodeling, the precise role of BK on VSMC structure and the resultant fibrosis has not been well studied. In the present study we have demonstrated that
BK induces the expression and transcriptional activation of ␣2(I) collagen gene in VSMC. In addition, we
have also shown that BK stimulates the production
and secretion rate of TIMP-1. The increase in ␣2(I)
collagen mRNA levels, ␣2(I) collagen promoter activity,
and TIMP-1 production in response to BK are mediated
via autocrine activation of TGF-␤1. Moreover, the increase in ␣2(I) collagen promoter activity, TGF-␤1, and
TIMP-1 production by BK involves activation of the
MAPK pathway. These findings provide the first evidence that BK stimulates several key signaling pathways that participate in matrix dysregulation in
VSMC.
Type I collagen represents up to two-thirds of total
collagen, and total collagen constitutes about 60% of
total atherosclerotic plaque protein composition (26,
37). Collagen type I is a heterotrimer consisting of two
␣1(I) chains and one ␣2(I) chain, which are expressed in
a coordinated manner. The expression of type I collagen is strictly regulated through development and is
tissue specific (31). In the present study, we have
shown that BK stimulates the mRNA levels of ␣2(I)
collagen in VSMC. To begin to understand the mechanism of this collagen regulation by BK, we examined
the effects of BK on VSMC transfected with the ␣2(I)
collagen promoter. Our results demonstrate that BK
stimulates the ␣2(I) collagen promoter activity, indicating that the increase in ␣2(I) collagen mRNA levels in
response to BK are mediated via transcriptional regulation. The ␣2(I) collagen promoter is regulated by
transcription factors such as SP1, SP3, CBF, and AP-1
(7, 41). We have previously shown that BK can induce
the expression of protooncogene c-fos and c-jun, and
the formation of AP-1 complex transcription factor
(15). The increase in c-fos mRNA levels and the formation of AP-1 complex in response to BK were mediated
via activation of p42mapk and p44mapk pathway (11, 28).
In this regard, our data demonstrate that activation of
p42mapk and p44mapk plays a critical role in mediating
the effects of BK on collagen regulation. Thus inhibition of the MAPK pathway significantly reduced the
increase in ␣2(I) collagen promoter activity in response
to BK. Therefore, one can speculate that activation of
BK receptors in VSMC in response to BK results in
activation of the MAPK pathway, which in turn results
in the formation of the transcription factor AP-1, which
leads to transcriptional activation of ␣2(I) collagen
gene.
TGF-␤ is the most potent and consistent activator of
collagen synthesis in VSMC (9). It increases the level of
steady-state mRNA of ␣2(I) and ␣1(III) collagen in
VSMC (1). In models of atherosclerosis such as rat
balloon injury, TGF-␤ was implicated as the factor
responsible for the increase in collagen production.
Transfection of TGF-␤ gene into pig artery results in
enhanced collagen type I production and accumulation
(27). In the model of rat balloon injury, the injection of
recombinant soluble TGF-␤ type II receptor caused a
marked reduction in mRNA levels for collagen type I
and III, thus indicating the role of TGF-␤ type II
receptor in the fibrotic activity of TGF-␤ (38). Similarly, in VSMC, a dominant-negative mutation of
TGF-␤ type II receptor abrogated the enhanced production of collagen type I induced by TGF-␤ (46). However,
the response of VSMC to TGF-␤ can be different ac-
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Fig. 7. Role of MAPK in BK-induced ␣2(I)
collagen promoter activity and TIMP-1
production in VSMC. A: VSMC were
transfected with a plasmid containing
␣2(I) collagen promoter and CAT gene as a
reporter for the activity of the ␣2(I) collagen promoter. Transfected cells were stimulated for 24 h with 10⫺8 M BK in presence or absence of MEK inhibitor
PD-98059 (40 ␮M). Bar graph represents
the ␣2(I) collagen promoter CAT activity
expressed as percent of control and is representative of 5 experiments. B: VSMC
were stimulated with 10⫺8 M BK in presence or absence of MEK inhibitor
PD-98059 (40 ␮M). Conditioned media
were collected and resolved on SDS-PAGE
and immunoblotted with a polyclonal antibody against rat TIMP-1. Bar graph represents intensities of TIMP-1 bands expressed as percentage of control. Blot is
representative of 4 separate experiments.
*P ⬍ 0.05 vs. control. †P ⬍ 0.05 vs. BK.
REGULATION OF VSMC FIBROSIS BY BK
retinoic acid is also mediated via activation of p42mapk
and p44mapk (3).
The role of MAPK in vascular dysfunction is just
beginning to be emphasized. The p42mapk and p44mapk
members of the MAPK family belong to a group of
serine/threonine kinases that are rapidly activated in
response to growth factor stimulation. They integrate
multiple signals from various second messengers leading to cellular proliferation and differentiation (8, 40).
The activated MAPK can translocate to the nucleus,
where it is thought to regulate the expression of transcription factors such as c-fos through the phosphorylation of the transcription factor p62TCF and the formation of AP-1 binding site (12). Recently, it has been
shown that MAPK activity is transiently activated
following vessel wall injury, whereas MAPK phosphatase-1 is decreased (18, 19). In a model of myocardial
infarction, MAPK activity was increased, along with
the mRNA levels of TGF-␤ and collagen types I and III
(35). In addition, NIH 3T3 cells transfected with a
dominant-negative mutant for MAPK kinase reduced
the increase in collagen type I promoter activity in
response TGF-␤ (25). In the present study we have
shown that BK mediates its fibrotic signals in VSMC
via activation of the MAPK pathway. Inhibition of
MAPK reduced the increase in ␣2(I) collagen promoter
activity, TGF-␤1 protein levels, and production of
TIMP-1 in response to BK. Other studies also support
a role for MAPK in modulating the effects of TGF-␤ on
fibronectin production in VSMC (16).
In summary, our results suggest that BK initiate
multiple signals that can result in fibrosis of VSMC.
Our findings demonstrate that treatment of VSMC
with BK results in an increase in ␣2(I) collagen mRNA
levels and promoter activity and an increase in the
production of TIMP-1. The increase in ␣2(I) collagen
promoter activity and TIMP-1 production in response
to BK are mediated via autocrine activation of TGF-␤
and via activation of the MAPK pathway. The significance of these findings to the in vivo actions of BK and
the vascular wall changes associated with injury or
atherosclerosis is unclear at the present time. Further
understanding of the cellular and molecular mechanisms by which BK might modulate vascular fibrosis, a
process obligatory to the development of atherosclerosis, could lead to the development of new strategies for
intervention and treatment of vascular diseases.
This work was supported by National of Institutes of Health
Grants HL-55782 and DK-46543, a Research Award from the American Diabetes Association (to A. A. Jaffa), a Merit Review Grant from
the Research Service of the Department of Veterans Affairs (to R. K.
Mayfield), a postdoctoral Award from the Juvenile Diabetes Foundation International (to V. Velarde), by National Heart, Lung, and
Blood Institute Training Fellowship HL-07260 (to J. T. Christopher),
and by a postdoctoral fellowship award from the Medical University
of South Carolina (to C. D. Douillet).
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