1 Department of Medicine and 2 Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina and Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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Vascular smooth muscle cell (VSMC) proliferation is a prominent
feature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. The
contribution of this system to vascular disease is undefined. In the
present study we characterized the signal transduction pathway leading
to mitogen-activated protein kinase (MAPK) activation in response to
bradykinin (BK) in VSMC. Addition of
1010-10
7
M BK to VSMC resulted in a rapid and concentration-dependent increase
in tyrosine phosphorylation of several 144- to 40-kDa proteins. This
effect of BK was abolished by the
B2-kinin receptor antagonist
HOE-140, but not by the B1-kinin
receptor antagonist des-Arg9-Leu8-BK.
Immunoprecipitation with anti-phosphotyrosine antibodies followed by
immunoblot revealed that
10
9 M BK induced tyrosine
phosphorylation of focal adhesion kinase (p125FAK). BK
(10
8 M) promoted the
association of p60src with the
adapter protein growth factor receptor binding protein-2 and also
induced a significant increase in MAPK activity. Pertussis and cholera
toxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C,
p60src kinase, and MAPK kinase
inhibited BK-induced MAPK tyrosine phosphorylation. These findings
provide evidence that activation of the
B2-kinin receptor in VSMC leads to
generation of multiple second messengers that converge to activate
MAPK. The activation of this crucial kinase by BK provides a strong
rationale to investigate the mitogenic actions of BK on VSMC
proliferation in disease states of vascular injury.
B2-kinin receptors; G protein receptors; tyrosine phosphorylation; signal transduction; mitogen-ativated protein kinase
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INTRODUCTION |
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VASCULAR INJURY IS considered to be a primary event in the evolution of atherosclerotic vascular disease. The "response-to-injury" hypothesis of atherosclerosis proposes that injury to the endothelium is the initiating event in atherogenesis (31). Support for endothelial injury or dysfunction as the initial lesion in atherosclerosis comes from experimental models of endothelial damage in which there is a resultant proliferation of the underlying smooth muscle cells (5, 6). Exaggerated proliferation of vascular smooth muscle cells (VSMC) is a characteristic finding in progressing atherosclerotic lesions (17).
Recent evidence indicates that abnormal synthesis and release of the vasodilator nitric oxide may contribute to vascular complications (22). Furthermore, nitric oxide can directly inhibit VSMC proliferation (14). Bradykinin (BK) is one of the most potent inducers of endothelial nitric oxide release (40). Indeed, BK causes relaxation of VSMC through synthesis and release of nitric oxide from the endothelium (39). In contrast, injury to the integrity of the endothelium enables BK to directly increase intracellular calcium levels and induce VSMC contraction (2). Exposure of VSMC to BK will occur through access from the circulation when the endothelium is injured or by generation within the vessel wall itself. A complete kallikrein-kinin system exists within the vascular wall. Kallikrein and its mRNA are expressed in isolated arteries and veins and in cultured VSMC (27, 28, 32). Kininogen, the substrate for kinin generation by kallikrein, kininase activity, and B2-kinin receptors are present in VSMC (9, 28). Thus locally generated kinins could act in an autocrine or paracrine fashion to influence vascular function.
Mitogen-activated protein kinases (MAPKs) belong to the group of serine-threonine kinases that are rapidly activated in response to growth factor stimulation. In mammalian cells the MAPK family includes extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2 or p42mapk and p44mapk), the c-Jun NH2-terminal kinases (JNK, also known as stress-activated protein kinase, SAPK), and p38mapk (7). They integrate multiple signals from various second messengers, leading to cellular proliferation or differentiation. 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 (15, 30, 38).
Although recent in vivo studies have linked the MAPK pathway to neointimal proliferation in response to arterial injury, the factor(s) responsible for this activation of MAPK is still undefined (21). A role for BK in the regulation of MAPK activity in VSMC has not been fully explored. Therefore, in the present study we examined whether BK can result in MAPK activation in VSMC and characterized the signal transduction pathway through which BK stimulates MAPK phosphorylation in VSMC. We provide evidence that BK via activation of B2-kinin receptors induces tyrosine phosphorylation of p42mapk, p44mapk, and focal adhesion kinase (p125FAK) and the association of p60src with growth factor receptor binding protein-2 (Grb2). The activation of MAPK by BK seems to involve a role for cytoplasmic tyrosine kinases and protein kinase C (PKC). These findings provide cellular and molecular evidence for a potential role for BK in VSMC function.
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METHODS |
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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 (24). A 2-cm segment of artery cleaned of fat and adventitia was incubated in 1 mg/ml collagenase for 3 h at room temperature. The artery was then cut into small sections and fixed to a culture flask for explantation in minimal essential medium containing 10% FCS, 1% nonessential amino acids, 100 mU/ml penicillin, and 100 µg/ml streptomycin. 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 trypsin-EDTA. VSMC were identified by the following criteria: they stained positive for intracellular cytoskeletal fibrils of actin and smooth muscle cell-specific myosin and negative for factor VIII antigens. VSMC isolated by this procedure were homogeneous and were used in all studies between passages 2 and 6.
Immunoprecipitation and immunoblotting.
VSMC were grown in serum-free medium for 24 h to render them quiescent.
Quiescent VSMC grown to subconfluence were stimulated with
1010-10
7
M BK for 5 min. The cells were then suspended in 250 µl of lysis buffer [20 mM Tris, 130 mM NaCl, 10% glycerol, 10 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium vanadate, 100 mU/ml aprotinin, 0.15 mg/ml benzamidine, pH 8.0], sonicated for
10 s, and centrifuged at 13,000 rpm for 10 min. Soluble protein (250-500 µg) obtained as described above was preincubated for 5 min with 10 µl of 10% Pansorbin. The suspension was centrifuged for
4 min at 10,000 rpm, and 500 µl of immunoprecipitation buffer (1.5%
Triton X-100, 150 mM NaCl, 10 mM Tris, 1 mM EGTA, 0.5% NP-40, 0.2 mM
sodium vanadate, 0.2 mM PMSF, pH 7.4) and 5 µg of the appropriate antibody were added to the resulting supernatant, which was incubated overnight at 4°C. The immunocomplex was recovered by incubation with 50 µl of Pansorbin at room temperature for 30 min, then
centrifugation at 10,000 rpm for 4 min. The pellet containing the
immunocomplex was resuspended in 20 µl of SDS sample buffer, boiled
for 5 min, and centrifuged, and the supernatant was analyzed by
SDS-PAGE. The separated proteins were transferred to polyvinylidine
difluoride membranes and immunoblotted with the desired antibody. The
immunoblot bands were visualized using enhanced chemiluminescence
reagent (Amersham) according to the procedure described by the supplier.
MAPK assay.
Quiescent VSMC grown to subconfluence were stimulated with
109-10
6
M BK for various times (0-30 min). At the desired time point the
incubation was terminated by addition of 0.5 ml of ice-cold PBS, and
the cells were scraped and pelleted by centrifugation for 30 s. The
pellet was resuspended in lysis buffer (20 mM HEPES, 80 mM
-glycerophosphate, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol), disrupted by sonication for 5 s, and centrifuged at 200,000 g for 20 min at 4°C to separate
the soluble and particulate fractions. MAPK activity in the cytosol was
assessed as the incorporation of
32P from
[
-32P]ATP into
myelin basic protein as substrate per microgram of cytoplasmic protein
(26).
Immunoblotting.
Quiescent VSMC stimulated with
107 M BK for 5 min were
suspended in 250 µl of lysis buffer (20 mM Tris, 130 mM NaCl, 10%
glycerol, 10 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM PMSF, 2 mM sodium vanadate, 100 mU/ml aprotinin, 0.15 mg/ml benzamidine, pH 8.0), sonicated for 10 s, and centrifuged at
13,000 g for 10 min. The cytosolic
fraction (25-30 µg) was analyzed by SDS-PAGE, and the separated
proteins were transferred to polyvinylidine difluoride membranes and
immunoblotted with anti-phospho-MAPK polyclonal antibody
(1:1,000 dilution; New England BioLabs, Beverly, MA).
Immunoreactive bands were visualized using the enhanced
chemiluminescence reagent according to the procedure described by the supplier.
Measurement of PKC activity in the cytosol and membrane fractions. VSMC grown in 150-mm plates were suspended in 0.5 ml of 10 mM Tris · HCl (pH 7.5) containing 0.25 sucrose, homogenized with a Polytron homogenizer for 5 s, and centrifuged at 3,000 rpm for 5 min. The supernatant was centrifuged at 19,000 rpm for 15 min, and the resultant supernatant was harvested as the cytosolic fraction. The membrane pellet fraction was resuspended in 50 µl of lysis buffer, incubated on ice for 30 min, and centrifuged at 12,000 g for 20 min. The supernatant will be retained as the membrane fraction. PKC activity in the cytosol and membrane fractions was measured by Western blots, as described above. The membranes were immunoblotted with antibodies against specific isoforms of PKC (1:3,000 dilution; Santa Cruz). The immunoreactive bands were visualized using the Renaissance chemiluminescence reagent, and the intensity of the bands was quantified by microdensitometer. Translocation of PKC from the cytosol to the membrane is indicative of activation.
Statistical analysis. Values are means ± SE and were analyzed by ANOVA. Differences were considered significant if P < 0.05.
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RESULTS |
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BK stimulates protein tyrosine phosphorylation in VSMC.
Protein phosphorylation reactions are essential for amplifying and
disseminating incoming signals throughout the cell. To examine the
pattern of tyrosine phosphorylation in response to BK, quiescent VSMC
were stimulated with
1010-10
7
M BK for 5 min. BK produced a concentration-dependent
increase in tyrosine phosphorylation of a number of 130- to 40-kDa
proteins, with a maximal response at
10
7 M (Fig.
1). The increase in tyrosine
phosphorylation of the 130- and 85-kDa bands by BK was quantified by a
densitometer, and the values are expressed as a percent increase
relative to control (for the 130-kDa band, 162 ± 21, 188 ± 37, 208 ± 38, and 213 ± 27 and for the 85-kDa band,
177 ± 47, 221 ± 52, 242 ± 42, and 241 ± 51 at
10
10,
10
9,
10
8, and
10
7 M BK, respectively,
P < 0.05). The increase in total
protein tyrosine phosphorylation induced by BK was blocked by the
B2-kinin receptor antagonist
HOE-140 (10
6 M), suggesting
that BK mediates its effects on tyrosine phosphorylation in VSMC via
activation of its B2 receptors
(Fig. 1).
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Activation of cytoplasmic tyrosine kinases by BK.
To investigate whether the 125-kDa protein band that was tyrosine
phosphorylated in response to BK is
p125FAK, quiescent VSMC were
stimulated with 109 M BK
for 5 min. The cell lysate was immunoprecipitated with
1 µg of anti-PY antibodies (Transduction Laboratories,
Lexington, KY) and immunoblotted with a specific
anti-p125FAK monoclonal antibody
(1:1,000 dilution; Transduction Laboratories). BK caused a significant
increase in the tyrosine phosphorylation of
p125FAK compared with unstimulated
cells (Fig. 2). Fetal bovine serum, used as
a positive control, also produced a twofold increase in tyrosine
phosphorylation of p125FAK.
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Activation of p42mapk and
p44mapk by BK.
The Erk family of MAPKs is rapidly activated in response to growth
stimuli and appears to integrate multiple intracellular signals
transmitted by various second messengers. To understand the signal
transduction events mediated by BK in VSMC, we examined its effects on
MAPK activation. Quiescent VSMC were treated with 106 M BK for various times
to determine optimal activation. MAPK activity was detectable within 1 min of BK addition, peaked at 5 min, and returned to near-basal levels
within 20-30 min (Fig. 3A): 581 ± 191, 612 ± 168, 1,682 ± 556, 638 ± 78, 774 ± 45, and 700 ± 274 pmol · min
1 · mg
protein
1 at 0, 1, 5, 10, 20, and 30 min, respectively. BK stimulated MAPK activation in a
concentration-dependent manner, with maximal activation at
10
7 M (Fig.
3B). Treatment with
10
8 M BK for 5 min also
resulted in a marked increase in tyrosine phosphorylation of
p44mapk and
p42mapk in VSMC, whereas minimal
phosphorylation was detected in unstimulated control cells (Fig.
3C).
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BK-induced phosphorylation of MAPK is mediated by the
B2-kinin receptor.
To determine the receptor subtype through which BK stimulates MAPK
phosphorylation, VSMC were pretreated for 30 min with the B1-kinin receptor antagonist
des-Arg9-Leu8-BK
(106 M) or the
B2-kinin receptor antagonist
HOE-140 (10
6 M), then
stimulated with 10
8 M BK
for 5 min. Treatment of VSMC with BK produced a fivefold increase in
p42mapk and
p44mapk phosphorylation compared
with unstimulated cells (Fig. 4). Addition of HOE-140 to VSMC prevented the BK-induced increase in MAPK
phosphorylation, whereas the
B1-kinin receptor antagonist had
no significant effect on BK-induced MAPK activity (Fig. 4). These
findings indicate that BK stimulates MAPK activity via the
constitutively expressed B2
receptors.
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Effects of pertussis and/or cholera toxin on BK-induced MAPK
phosphorylation.
To elucidate the mechanisms leading to MAPK activation in response to
BK, we examined the roles of various signaling molecules activated by
BK through its B2 receptor. The
B2-kinin receptor was reported to
be coupled to Gq,
G13, or
Gi and
Go, which activates phosphatidylinositol-specific phospholipase C and inhibits adenylate cyclase, respectively. To determine through which G protein BK signals
to stimulate MAPK phosphorylation, we studied the effect of pertussis
toxin (PTx), which inhibits Gi,
and cholera toxin (CTx), which downregulates
Gs, on BK-induced MAPK activation
in VSMC. Treatment of VSMC with PTx (100 ng/ml) for 24 h did not affect
BK-induced MAPK phosphorylation, whereas it completely blocked the
effect on MAPK phosphorylation of lysophosphatidic acid (LPA), a
well-characterized agonist that uses a
Gi-coupled receptor (2,685 ± 943, 8,621 ± 146, and 2,303 ± 117 densitometric units for
control, LPA, and LPA + PTx, respectively). Similarly, treatment with
CTx (5 µg/ml) also did not affect BK-induced MAPK phosphorylation,
indicating that BK activates MAPK via PTx- and CTx-insensitive G
proteins (Fig. 5).
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Role of PKC in BK-induced MAPK phosphorylation.
In VSMC, activation of phospholipase C by BK leads to production of two
second messengers, inositol phosphates and diacylglycerol, which induce
the release of intracellular calcium and PKC activation (8). To
evaluate whether PKC is essential for BK-induced MAPK activation, VSMC
were treated with 108 M BK
for 5 min in the presence of a PKC inhibitor, bisindolylmaleimide (2 µM), and/or after downregulation of PKC by 24 h of pretreatment with
phorbol ester [5 µM phorbol 12-myristate 13-acetate
(PMA)]. As shown in Fig. 6, PKC
depletion by PMA reduced BK-induced MAPK phosphorylation by >80%
compared with BK-treated cells. Treatment with the PKC inhibitor
bisindolylmaleimide also significantly reduced MAPK phosphorylation by
BK (Fig. 6). We have also studied the effects of another PKC inhibitor,
H7 (10 µM), on BK-induced MAPK
phosphorylation. We found that H7
also blocked BK-induced MAPK phosphorylation by ~60%: 870 ± 177 and 337 ± 97% of MAPK phosphorylation above control for BK and BK + H7, respectively (P < 0.05). Taken together, these
data indicate that BK activates MAPK via a PKC-dependent pathway.
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Role of cytoplasmic kinases in BK-induced MAPK phosphorylation.
To identify the cytoplasmic kinases involved in BK-induced MAPK
phosphorylation, we examined the effects of specific cell-permeable kinase inhibitors, such as PP1 (Biomol Research Laboratories, Plymouth,
PA), which inhibits the Src family tyrosine kinases; wortmannin (Sigma
Chemical, St. Louis, MO), which inhibits phosphatidylinositol 3-kinase
(PI3K); and PD-98059 (New England BioLabs), which specifically inhibits
the MAPK activator MAPK kinase (1, 9, 14). Quiescent VSMC were
pretreated with the PP1 inhibitor (10 µM) for 2 h and/or wortmannin
(10 nM) for 30 min and/or PD-98059 (40 µM) for 30 min, then
stimulated with 108 M BK
for 5 min. The results are shown in Fig. 8.
BK once again produced a four- to sixfold increase in the
phosphorylation of p42mapk and
p44mapk. This increase in MAPK
phosphorylation was completely blocked by the Src kinase inhibitor and
the MAPK kinase inhibitor but not by the PI3K inhibitor (Fig. 8).
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DISCUSSION |
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The signal transduction pathway through which BK activates MAPK in VSMC is still undefined. In the present study we have identified several second messenger systems that are generated on activation of the B2-kinin receptor in response to BK. We have shown that BK can induce tyrosyl phosphorylation of a number of proteins in VSMC. Immunoprecipitation and immunoblotting techniques revealed that BK can induce tyrosine phosphorylation of p125FAK and the association of p60src with the adapter protein Grb2. BK also induced the activation and tyrosyl phosphorylation of p42mapk and p44mapk. The tyrosine phosphorylation of p42mapk and p44mapk in VSMC by BK is PTx and CTx insensitive and involves the activation of PKC and cytoplasmic tyrosine kinases. These findings provide the first evidence that BK stimulates early mitogenic signals associated with activation of ERK in VSMC.
It is generally accepted that ERK activation plays a principal role in cell proliferation and differentiation (30, 38). In the present study we have shown that BK induces rapid activation of ERK in VSMC. Once activated, ERKs can translocate to the nucleus, thus providing a link in the signal transduction pathway from the cytoplasm to the nucleus. In this regard, we previously showed that BK results in activation and nuclear translocation of MAPK in mesangial cells (18). Once in the nucleus, ERK can phosphorylate and activate transcription factors such as TCF/ELK-1, resulting in c-Fos production and AP-1 complex formation (13, 15).
The initiating and sustaining signals that link kinin receptor activation to MAPK regulation are not defined. The B2-kinin receptor is a member of the seven-transmembrane G protein-coupled receptor superfamily (25). On binding to its receptors in VSMC, BK activates phospholipase C via a heterotrimeric GTP-binding protein and induces a marked increase in inositol 1,4,5-trisphosphate, an increase in intracellular calcium, and generation of diacylglycerol, ultimately resulting in PKC activation (8). Luttrel and co-workers (23) demonstrated that the mechanisms involved in MAPK activation are heterogeneous, depending on which receptor and cell type are under consideration. Thus MAPK activation by B2 receptors may occur via an Ras-dependent or -independent means by utilization of a PTx-sensitive or -insensitive G protein. In this regard, we have shown that BK, unlike LPA, stimulates MAPK via PTx- and CTx-insensitive mechanisms. This finding may suggest that the G protein Gq, which couples the B2 receptor, plays a dominant role in MAPK activation by BK, rather than Gi or Go.
Because Gq-coupled receptors have
been shown to activate MAPK via a PKC-dependent mechanism, we reasoned
that BK through activation of
B2-kinin receptor would stimulate
ERK activity via activation of PKC. In support of our notion, we
observed that pretreatment of VSMC with PMA to downregulate PKC
activity completely eliminated the increase in MAPK phosphorylation in
response to BK stimulation. In addition, we found that the PKC
inhibitor bisindolylmaleimide also significantly suppressed the
increase in MAPK phosphorylation induced by BK. Taken together, these
findings suggest that BK stimulates MAPK activation via a PKC-dependent
mechanism. The specific PKC isoform that is responsible for MAPK
activation by BK and/or the upstream second messengers that result in
PKC activation by BK are undefined. In this regard, our data indicate
that BK results in the activation of PKC-. Once activated, PKC-
has been shown to activate Raf-1 via phosphorylation, which in turn will result in MAPK activation (19, 20, 40). Not all
Gq-coupled receptors activate MAPK
through similar mechanisms. For instance, the ANG II receptor, which is
a Gq-coupled receptor, activates MAPK via a PKC-independent pathway, indicating that BK and ANG II
induce different signaling pathways that result in MAPK activation in
VSMC (12).
In the case of receptors with intrinsic tyrosine kinase activity, several steps in the signal transduction pathway leading to MAPK activation have been elucidated. Interaction of growth factors (e.g., insulin and insulin-like growth factor-I) with their respective receptors activates the intracellular tyrosine kinase domains, which results in the phosphorylation of Shc followed by association with the adapter protein Grb2, which in turn promotes the interaction of guanine nucleotide-releasing protein Sos with Ras, leading to its activation. Activated Ras causes activation of Raf-1, which phosphorylates and activates MAPK kinase, ultimately leading to phosphorylation and activation of MAPK (37).
The B2-kinin receptor lacks an intrinsic tyrosine kinase. However, when we treated VSMC with BK, we observed a rapid dose-dependent increase in tyrosine phosphorylation of a number of proteins. This increase in tyrosyl phosphorylation by BK was blocked by the B2-kinin receptor antagonist but not by the B1 receptor antagonist, indicating a B2 receptor-mediated event. The cellular mechanism(s) through which BK stimulates tyrosyl phosphorylation is undefined but alludes to the possibility of recruitment of a cytoplasmic tyrosine kinase by the activated receptor. Using immunoprecipitation and immunoblotting techniques, we determined that one of these proteins that is tyrosyl phosphorylated by BK is the cytoplasmic tyrosine kinase p125FAK. Tyrosine phosphorylation of p125FAK has been directly correlated with increased protein tyrosine activity and has been linked to signal transduction events through activation of G protein-coupled receptors and, on integrin binding, to extracellular matrix proteins (3, 29). Thus p125FAK could act as a point of convergence of multiple signals triggered by activation of different receptors (41). The cellular mechanism(s) by which p125FAK is phosphorylated are not clearly defined, but recent studies implicate a role for the Src family of tyrosine kinases. Transformation of cells with oncogenic variants of Src leads to an increase in tyrosine phosphorylation of p125FAK, which then forms a stable complex with Src kinase through interactions with the SH2 domains of Src (33, 34). Thus the phosphorylation of p125FAK and the interaction of Src with FAK may be prerequisites for Src activation and for subsequent sequential signaling events that promote tyrosine phosphorylation of other focal adhesion proteins and binding to the adapter protein Grb2 (35, 36). The adapter protein Grb2 binds to the tyrosine-phosphorylated sites on activated protein tyrosine kinases through its SH2 domain and complexes with the exchange factor Sos, through its SH3 domain, thus providing a network for signal transmission (11). In this regard, our findings in VSMC indicate that BK stimulation promotes the association of pp60src with the adapter protein Grb2. This binding of Src to Grb2 could in turn activate Ras, which would eventually result in p42mapk and p44mapk activation, thus providing one signaling mechanism by which BK can activate the MAPK pathway.
To further delineate the role of cytoplasmic tyrosine kinases in
BK-induced MAPK activation, we examined the effects of cell-permeable specific inhibitors of p60src:
PI3K and MAPK kinase. Pretreatment of VSMC with the PI3K inhibitor wortmannin did not alter MAPK activation by BK, suggesting that PI3K
does not contribute to BK-induced MAPK signaling. On the other hand,
the Src kinase inhibitor and/or the MAPK kinase inhibitor completely
eliminated the increase in MAPK phosphorylation induced by BK.
Moreover, the increase in PKC- activity by BK was eliminated when
Src kinase activity was inhibited, suggesting that Src kinase is
upstream of PKC. This finding lends further support for a role of Src
kinase in the signal transduction pathway leading to MAPK activation by BK.
In summary, we propose that activation of the B2 receptor by BK leads to the generation of multiple second messengers that converge to activate MAPK. The present findings provide cellular and molecular evidence for a potential role of BK in VSMC function. The significance of these findings to the in vivo actions of BK on the pathological progression initiated by vascular wall changes is unclear. Because the in vivo physiological actions of BK are modulated by various autacoids, such as eicosanoids and nitric oxide, the contribution of these second messenger systems to the signaling mechanisms initiated by BK in VSMC must be considered. Better understanding of the molecular and cellular mechanisms by which kinins stimulate VSMC proliferation in vivo, a process obligatory to the development of vascular injury, could lead to the development of new strategies for intervention and treatment of vascular diseases.
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ACKNOWLEDGEMENTS |
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We thank Kim Sutton for technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-46543 and 1-PO-1-HL-55782 and a Research Award from the American Diabetes Association (A. A. Jaffa), a Merit Review Grant from the Research Service of the Department of Veterans Affairs (R. K. Mayfield), and a Postdoctoral Research Fellowship Award from the Juvenile Diabetes Foundation (V. Velarde).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. A. Jaffa, Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: jaffaa{at}musc.edu).
Received 15 July 1998; accepted in final form 27 April 1999.
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