(Received for publication, June 7, 1995; and in revised form, August 25, 1995)
From the
It has recently been reported that protein-tyrosine kinase
activity is required for thrombin-induced growth in vascular smooth
muscle cells (VSMC). In the present study, we have identified several
phosphoproteins that are tyrosine-phosphorylated in response to
thrombin in quiescent VSMC. These proteins are insulin-like growth
factor-1 receptor -subunit (IGF-IR
), insulin receptor
substrate-1 (IRS-1), and phospholipase C-
1 (PLC-
1).
Thrombin-stimulated phosphorylation of these proteins was rapid; it was
maximal at 1 min and reduced thereafter. Thrombin also activated
mitogen-activated protein kinases (MAPK) in quiescent VSMC in a
biphasic manner with a rapid and larger peak at 10 min (6-fold)
followed by a sustained smaller second peak at 2 h (2-fold). Inhibition
of protein-tyrosine kinase activity by the use of two structurally
different protein-tyrosine kinase inhibitors, genistein and herbimycin
A, significantly blocked the thrombin-induced tyrosine phosphorylation
of IGF-1R
, IRS-1, and PLC-
1 and decreased thrombin-stimulated
DNA synthesis. In contrast, however, inhibition of protein-tyrosine
kinase activity had no effect on thrombin activation of MAPK.
Collectively, these findings suggest a role for tyrosine
phosphorylation of IGF-IR
, IRS-1, and PLC-
1 in
thrombin-induced mitogenic signaling events in VSMC. Furthermore, while
protein tyrosine phosphorylation is essential for thrombin-induced DNA
synthesis, it is not required for thrombin-stimulated MAPK activation.
Since thrombin rapidly activated Src in VSMC, Src may be involved in
the cross-talk between the G-protein-coupled receptor agonist and a
tyrosine kinase receptor such as IGF-1R.
Vascular smooth muscle cell (VSMC) ()growth
(hyperplasia and hypertrophy) is considered to be a significant factor
in atherosclerosis and restenosis following balloon
angioplasty(1) . Several molecules including platelet-derived
growth factor (PDGF-A and -B), fibroblast growth factor, and
angiotensin II appear to be involved in the induction of paracrine and
autocrine growth in VSMC(1) . More recently it has been
reported that thrombin, a serine protease, in addition to its role in
blood coagulation also stimulates growth in certain cell types, notably
fibroblasts and VSMC(2, 3) . In further support of an
important role of thrombin in this vascular lesion formation, it has
been documented that hirudin, a specific inhibitor of thrombin, reduces
neointimal formation(4, 5) .
Thrombin is thought to transmit both its coagulant and mitogenic effects via activation of its receptor, a transmembrane G-protein-coupled receptor, which is identical with or similar to the one we and others recently cloned(6, 7) . The observation that hirudin but not the anticoagulant heparin inhibited formation of a neointima suggests that the coagulant and mitogenic effects of thrombin are regulated by differential mechanisms or that there may be more than one type of thrombin receptor(5) . In fact, using thrombin receptor-activating peptides and protein-tyrosine kinase inhibitors some investigators have demonstrated that along with activation of its G-protein-coupled receptor, thrombin also requires protein-tyrosine kinase activity, possibly of a receptor type for its mitogenic effect (2, 3) .
Mitogen-activated protein kinases (MAPK) are implicated in the transmission of growth signals from activated receptor tyrosine kinases and G-protein-coupled receptors(8, 9) . MAPK are a family of serine/threonine kinases, which are activated by phosphorylation both at threonine and tyrosine residues. These reactions are catalyzed by a dual specificity enzyme MAPK kinase, also termed MEK(10, 11) . MEK itself is activated by at least two types of kinases, Raf-1(12, 13) and MEK kinase, also known as MEKK(14) . Raf-1, which is also activated by phosphorylation, appears to be central in receiving signals from activated upstream kinases such as receptor tyrosine kinases(9, 15) , protein kinase C(16) , and G-protein-coupled receptors(17, 18) . This serine/threonine kinase pathway is thought to be involved in the convergence of growth-initiated early protein-tyrosine kinase signaling events and the subsequent transduction of these signals into the nucleus(19) .
Since thrombin is an effector of several
important cellular processes and is active in many pathological
conditions, it is important to understand precisely the signaling
events by which this protease deciphers its diverse biological effects.
Therefore, the goal of this study was to investigate signaling events,
particularly the role of protein tyrosine phosphorylation and MAPK
activation in thrombin-induced growth of VSMC. We report several novel
observations. First, thrombin stimulates VSMC DNA synthesis, and
tyrosine phosphorylation of several proteins, including IGF-1R, IRS-1,
and PLC-1, is associated with this phenomena. Second, thrombin
activated MAPK in a biphasic manner in quiescent VSMC. Third,
inhibition of protein-tyrosine kinase activity blocked the
thrombin-induced tyrosine phosphorylation of IGF-1R, IRS-1, and
PLC-
1 and DNA synthesis but not thrombin-induced MAPK activation,
suggesting that the activity of MAPK is dissociated from early
protein-tyrosine kinase events induced by thrombin in VSMC. Fourth,
thrombin activated Src rapidly in quiescent VSMC, a result that
suggests a possible role for this nonreceptor type protein-tyrosine
kinase in the cross-talk between the thrombin-bound G-protein-coupled
receptor and a protein-tyrosine kinase receptor such as IGF-1R.
Growth-arrested VSMC were treated with various concentrations
of thrombin (0.025-0.2 unit/ml) for 24 h, and DNA synthesis was
measured by [H]thymidine incorporation. Thrombin
stimulated VSMC DNA synthesis in a dose-dependent manner (Fig. 1). Increases in VSMC DNA synthesis were caused by
thrombin at a concentration as low as 0.025 unit/ml. A maximum 20-fold
increase in VSMC DNA synthesis was observed with 0.1 unit/ml thrombin,
a result that indicates that thrombin is capable of causing growth in
VSMC. To determine whether thrombin-stimulated DNA synthesis requires
protein-tyrosine kinase activity, growth-arrested VSMC were treated
with thrombin (0.1 unit/ml) in the presence and absence of 25
µM genistein and/or 1 µM herbimycin A, two
structurally different but potent protein-tyrosine kinase
inhibitors(23, 24) , and DNA synthesis was measured.
Genistein inhibits receptor and non-receptor type protein-tyrosine
kinase activity by competing with ATP(25) , while herbimycin A
inhibits cellular protein-tyrosine kinase activities, such as Src
irreversibly by a mechanism involving interactions of benzoquinone with
protein sulfhydryl groups(26) . As shown in Fig. 2, both
genistein and herbimycin A significantly blocked thrombin-induced DNA
synthesis. Genistein and herbimycin A toxicity was determined by
measuring cell viability by trypan blue dye exclusion assay 48 h after
exposing VSMC to these compounds. At the concentrations used in these
studies, these compounds were not toxic. Higher concentrations (50
µM genistein and 10 µM herbimycin A) were
found to be toxic to VSMC with a longer incubation period. Therefore,
inhibition of DNA synthesis by genistein and herbimycin A suggests that
protein-tyrosine kinase activity is required for thrombin-induced
growth in VSMC.
Figure 1:
Thrombin stimulates VSMC DNA synthesis
in a dose-dependent manner. Growth-arrested VSMC were treated with and
without the indicated concentrations of thrombin for 24 h, and DNA
synthesis was measured by [H]thymidine
incorporation into acid-precipitable material as described under
``Experimental Procedures.'' Values are mean ± S.D. of
three separate experiments performed in triplicate each
time.
Figure 2: Protein-tyrosine kinase inhibitors attenuate thrombin-induced DNA synthesis in VSMC. Growth-arrested VSMC were treated with and without thrombin (0.1 unit/ml) in the presence and absence of herbimycin A (1 µM) or genistein (25 µM) for 24 h, and DNA synthesis was measured as described in the legend for Fig. 1. Values are mean ± S.D. of three separate experiments performed in triplicate each time.
Many studies have shown that thrombin stimulates
tyrosine phosphorylation of several proteins including Src in various
cell types (27, 28, 29, 30) . To
identify the proteins that were potentially tyrosine-phosphorylated by,
and involved in, thrombin-induced mitogenic signaling, growth-arrested
VSMC were treated with thrombin (0.1 unit/ml) for various time periods,
and cell extracts were prepared. Extracts of thrombin-treated and
untreated VSMC containing equal amounts of proteins (100 µg) were
analyzed by immunoblotting with phosphotyrosine antibodies. We detected
three phosphoproteins with apparent molecular masses of 100, 140, and
180 kDa, whose tyrosine phosphorylation in response to thrombin
increased rapidly (Fig. 3). Maximum 2-3-fold increases in
the phosphotyrosine content of these proteins were observed at 1 min of
thrombin treatment, gradually decreasing thereafter. The apparent
molecular masses of these three phosphoproteins (100, 140, and 180 kDa)
appeared to fall within the range of molecular masses reported for
IGF-IR, PLC-
1, and IRS-1,
respectively(31, 32) . To establish the identity of
these proteins, equal amounts of proteins (500 µg) from
thrombin-treated and untreated VSMC were immunoprecipitated with
anti-IGF-IR
, anti-PLC-
1, or anti-IRS-1 antibodies and
analyzed by Western blotting using phosphotyrosine antibodies.
Phosphotyrosine Western blot analysis of anti-IGF-IR
immunoprecipitates detected a band with a molecular mass of 100 kDa,
whose phosphorylation was increased severalfold in response to
thrombin, and this response was sensitive to inhibition by genistein (Fig. 4). Phosphotyrosine Western blot analysis of anti-IRS-1
immunoprecipitates showed a band of 180 kDa in size, and its
phosphotyrosine content was increased 2.5-fold by thrombin (Fig. 4). Genistein inhibited the thrombin's effect on
IRS-1 phosphorylation. The identity of the 140-kDa protein as
PLC-
1 was also established using a similar approach (Fig. 4). It has recently been reported that Src phosphorylates
IGF-1R in Rat-1 fibroblasts(33) . Therefore, to investigate a
possible mechanism by which thrombin stimulates IGF-1R phosphorylation,
thrombin's effect of Src activation in growth-arrested VSMC was
determined. As shown in Fig. 5, thrombin activated Src rapidly
in VSMC with a maximum effect at 1 min (3-fold). In addition, thrombin
activation of Src was found to be sensitive to protein-tyrosine kinase
inhibition (Fig. 5). Collectively these findings strongly
suggest a role for tyrosine phosphorylation of IGF-1R, IRS-1,
PLC-
1, and Src in thrombin-initiated mitogenic signaling events.
Figure 3: Thrombin stimulates protein tyrosine phosphorylation in VSMC. Growth-arrested VSMC were treated with and without thrombin (0.1 unit/ml) for the indicated time periods, and cell extracts were prepared. One-hundred micrograms of protein from each treatment was resolved on SDS-PAGE and analyzed for phosphotyrosine proteins by Western blotting using phosphotyrosine antibodies. Size markers in kDa are shown on the right-hand side.
Figure 4:
Thrombin induces tyrosine phosphorylation
of IGF-1R, IRS-1, and PLC-1, and protein-tyrosine kinase
inhibitors abrogate these responses. Growth-arrested VSMC were treated
with and without thrombin (0.1 unit/ml) in the presence and absence of
herbimycin A (H) (1 µM) or genistein (G)
(25 µM) for the indicated time periods, and cell extracts
were prepared. Five hundred micrograms of protein from each treatment
was incubated with IGF-1R
, IRS-1, or PLC
-1 antibodies
overnight, and the immunoprecipitates were Western blot analyzed using
phosphotyrosine antibodies.
Figure 5: Thrombin activation of Src. Growth-arrested VSMC were treated with and without thrombin (0.1 unit/ml) in the presence and absence of genistein (G) (25 µM) for the indicated time periods, and cell extracts were prepared. Equal amounts of trichloroacetic acid-precipitable counts/min from each treatment were immunoprecipitated with monoclonal Src antibodies, and the immunocomplexes were resolved by SDS-PAGE.
It was thought that early protein tyrosine phosphorylation events
such as receptor tyrosine kinase phosphorylation transmit mitogenic
signals by activation of a ``Ras-Raf-MEK-MAPK'' pathway via
recruitment of adaptor molecules such as GRB2, Shc, and
SOS(19, 34, 35, 36) . Since thrombin
requires protein-tyrosine kinase activity for its mitogenic effect and
IGF-IR phosphorylation is associated with this, it is paradoxical and
intriguing that these tyrosine phosphorylation events may be required
for thrombin's activation of the ``Ras-Raf-MEK-MAPK''
pathway. Several experiments were performed to test this hypothesis.
Growth-arrested VSMC were treated with thrombin (0.1 unit/ml) for
various time periods as well as in the presence and absence of
protein-tyrosine kinase activity inhibitors, and cell lysates were
prepared. MAPK activities in cell lysates were determined by 1) in-gel
kinase assay using MBP as a substrate and 2) detection by mobility
shift of phosphorylated MAPK on Western blots. Cell lysates containing
equal amounts of proteins from control and various treatments were
resolved by SDS-PAGE copolymerized with MBP, and the kinase assay was
performed by incubating the gel in the kinase buffer containing
[P]ATP, as described under
``Experimental Procedures.'' Thrombin activated MAPK in a
time-dependent biphasic manner with the first and highest peak of
activity (6-fold) at 10 min, followed by a second and more sustained
lower peak of activity (2-fold) at 2 h (Fig. 6). These results
are in agreement with previous reports in hamster
fibroblasts(37) . Genistein or herbimycin A alone had no effect
on MAPK activities in VSMC and did not block the thrombin-stimulated
activation of MAPK in these cells (Fig. 6). The lack of the
effect of genistein on thrombin-stimulated activation of MAPK is
selective, because it blocked the serum-induced activation of MAPK in
VSMC (Fig. 7). In fact, Winitz et al.(18) and
Hawes et al.(38) also observed that genistein, a
protein-tyrosine kinase inhibitor, did not block the muscarinic
cholinergic receptor, a G-protein-coupled receptor, mediated activation
of MAPK, while it inhibited epidermal growth factor receptor-mediated
activation of MAPK.
Figure 6: Thrombin activates MAPK in a biphasic manner, and these responses are insensitive to inhibition by protein-tyrosine kinase inhibitors. Growth-arrested VSMC were treated with and without thrombin (0.1 unit/ml) in the presence and absence of herbimycin A (H) (1 µM) or genistein (G) (25 µM) for the indicated time periods, and cell extracts were prepared. Fifty micrograms of protein were resolved on mini-SDS-PAGE that was copolymerized with myelin basic protein. MAPK activities were measured by in-gel kinase assay as described under ``Experimental Procedures.''
Figure 7: Genistein blocks the serum-induced activation of MAPK. Growth-arrested VSMC were treated with and without serum (10%, v/v) in the presence and absence of genistein (G) (25 µM) for 5 min, and cell extracts were prepared. MAPK activity was measured as described in the legend for Fig. 6.
These findings provide the first demonstration
that thrombin, a G-protein-coupled receptor agonist, stimulates
tyrosine phosphorylation of IGF-1R, a receptor protein-tyrosine kinase.
IGF-1R consists of two (135 kDa) and two
(95 kDa) subunits
linked by disulfide bonds. Ligand binding to the extracellular
-subunit of the receptor produces a conformational change in the
-subunits resulting in autophosphorylation, which is required for
its kinase activity. In turn the activated receptor phosphorylates
several proteins including its immediate substrate IRS-1(39) .
Phosphorylated IRS-1 interacts with downstream signaling molecules such
as GRB2 and phosphatidylinositol 3-kinase via Src homology 2 (SH2)
domains(40, 41) . IGF-1R has a critical role in
transmitting its ligand-evoked cellular
effects(19, 42) , and the importance of IGF-1R
phosphorylation in cell growth and transformation has also been
demonstrated(39, 43) . In fact, Peterson et al.(33) recently reported ligand-independent phosphorylation
of IGF-1R in Src-transformed fibroblasts. This work also showed that
Src-induced intracellular phosphorylation events are required for
IGF-1R phosphorylation in these cells. A similar mechanism may account
for thrombin-induced phosphorylation of IGF-1R in VSMC. This suggestion
is based on the following observations. 1) Thrombin activated Src in
VSMC, and 2) this event is temporally correlated with phosphorylation
of IGF-1R by thrombin. As thrombin-induced early protein tyrosine
phosphorylation events include phosphorylation of Src, IGF-1R, IRS-1,
and PLC-
1 and inhibition of protein-tyrosine kinase activity
blocks thrombin's phosphorylation of these molecules and DNA
synthesis, it is likely that thrombin-induced Src, IGF-1R, IRS-1, and
PLC-
1 phosphorylation are closely associated with thrombin's
mitogenic signaling events in VSMC.
Several investigators have
established that ligand-induced receptor tyrosine kinase activation is
essential for MAPK
activation(19, 34, 35, 36, 40, 41, 42) .
MAPK are important in regulating transcriptional factors such as
c-jun, c-myc, and
p62(44, 45, 46) , and they in
turn modulate gene transcription. Although our findings demonstrate a
biphasic activation of MAPK by thrombin this event appears to be
dissociated from tyrosine phosphorylation of IGF-1R, IRS-1, PLC-
1,
and Src, since inhibiting phosphorylation of these molecules had no
effect on thrombin-induced activation of MAPK. On the other hand,
induced tyrosine phosphorylation of IGF-1R, IRS-1, PLC-
1, and Src
appears to be on the pathway that leads to DNA synthesis because
inhibiting protein-tyrosine kinase activity blocked phosphorylation of
these molecules as well as DNA synthesis. Pages et al.(47) by antisense down-regulation of MAPK and by
overexpression of MAPK reported that MAPK are required for fibroblast
growth factor and thrombin-induced fibroblastic growth. Our findings
and those of Pages et al.(47) imply that the tyrosine
phosphorylation events that we observed and the MAPK cascade are
independently regulated by thrombin, and both are required for DNA
synthesis. If both the MAPK pathway and protein-tyrosine kinase events
are required for DNA synthesis, then interference with either pathway
will affect growth. Another explanation is that both thrombin-induced
IGF-1R, IRS-1, PLC-
, and Src tyrosine phosphorylation and MAPK
activation events may converge downstream to inhibition by
protein-tyrosine kinase inhibitors. Thrombin-stimulated MAPK activation
may be mediated by a mechanism involving protein kinase C. In fact,
thrombin has been shown to activate protein kinase C in other cell
types (48) and angiotensin II, a G-protein-coupled receptor
agonist, reportedly activated MAPK by a protein kinase C-dependent
mechanism in VSMC(49) . Future studies are required to
elucidate the requirement for MAPK activation for thrombin-induced DNA
synthesis and whether serine/threonine kinases interact with protein
tyrosine phosphorylation events to cause DNA synthesis in VSMC.