(Received for publication, December 7, 1995; and in revised form, January 29, 1996)
From the
Activation of adrenergic receptors stimulates
mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine
signaling pathways by which activation of
receptors
may induce mitogenesis in HVSMCs, we have found that
receptor stimulated-DNA synthesis and activation of
mitogen-activated protein (MAP) kinase are blocked by wortmannin, an
inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine
directly if activation of
receptors stimulated PI
3-kinase, in vitro assays of kinase activity were performed in
immunocomplexes precipitated by an antibody against the p85
subunit of PI 3-kinase. Noradrenaline stimulated a time- and
concentration-dependent activation of PI 3-kinase in the presence of a
adrenergic receptor antagonist. Noradrenaline-stimulated PI
3-kinase activation was blocked by antagonists of
receptors and by pertussis toxin, suggesting that
receptors activate PI 3-kinase via a pertussis toxin-sensitive G
protein. Direct activation of protein kinase C by a phorbol ester did
not stimulate PI 3-kinase; also, a Ca
L-channel
blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity.
Increased PI 3-kinase activity was detected in both anti-Ras and
anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated
HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP
complexes. Because blockade of PI 3-kinase by wortmannin inhibited
formation of this complex, this result suggests that Ras might be a
target of PI 3-kinase. Noradrenaline stimulated tyrosine
phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated
tyrosine protein could be co-immunoprecipitated with anti-p85 of PI
3-kinase. These results demonstrate that stimulation of
receptors activates PI 3-kinase in HVSMCs and that
receptor-activated PI 3-kinase is associated with an increase in
active Ras-GTP and activation of tyrosine protein phosphorylation.
These pathways may contribute to
receptor-stimulated
mitogenic responses including activation of MAP kinase and DNA
synthesis in HVSMCs.
adrenergic receptors are members of the
superfamily of G protein-coupled membrane receptors; these pathways
mediate many of the important physiological effects of catecholamines
such as noradrenaline and adrenaline.
adrenergic
receptors play a particularly important role in control of blood
pressure via induction of vascular smooth muscle contraction (Minneman
and Esbenshade, 1994). Also, activation of
adrenergic
receptors stimulates cardiac and vascular smooth muscle growth and
hypertrophy (Milano et al., 1994; Nakafuku et al.,
1990; and Okazaki et al., 1994). However, signaling pathways
utilized by
receptors in promoting mitogenic effects,
such as growth-related gene expression and DNA synthesis, are unclear.
It is generally accepted that activation of receptors stimulates phospholipase C, leading to increased
hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol
1,4,5-trisphosphate and 1,2-diacylglycerol. Both inositol
1,4,5-trisphosphate and 1,2-diacylglycerol play important roles as
intracellular second messengers that increase intracellular
Ca
concentrations and activate various isoforms of
protein kinase C, respectively. These coupling mechanisms are typically
mediated by pertussis toxin-insensitive G proteins, likely in the Gq/11
family (Perez et al., 1993; Schwinn et al., 1995).
Additionally, stimulation of
receptors activates
phospholipase D and phospholipase A2 via pertussis
toxin-insensitive/sensitive G proteins (Minneman and Esbenshade, 1994;
Perez et al., 1993).
Although this predominant view of
receptor signaling provides substantial insight into
receptor-mediated responses in various cells, there
are clear indications that these mechanisms may not explain all aspects
of
receptor signaling. For example, recent evidence
demonstrates that
receptor-stimulated mitogenic
responses in myocytes may be due to activation of tyrosine protein
kinases (TPKs) (
)and MAP kinases (Thorburn et al.,
1994), suggesting that
adrenergic receptors may share
common signal pathways with tyrosine kinase receptors in the
stimulation of mitogenesis.
There has been considerable recent
interest in lipid kinases that phosphorylate the 3-position of the
inositol ring of inositol phospholipids; this has led to the
identification of the enzyme PI 3-kinase (for reviews see Carpenter and
Cantley(1990), Divecha and Irvine(1995), Fry(1994), and Valius and
Kazlauskas(1993). PI 3-kinase is a lipid kinase that has been
implicated in the regulation of cell growth and proliferation by
receptor tyrosine kinases (Ruderman et al., 1990; Valius and
Kazlauskas, 1993), nonreceptor tyrosine kinases (Ding et al.,
1995), cytokine receptors (Karnitz et al., 1995) and oncogene
products (Fukui et al., 1991). Stimulation of cells with
mitogens such as platelet-derived growth factor and many other peptide
growth factors leads to accumulation of the lipid products
phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol
3,4,5-trisphosphate (Carpenter and Cantley, 1990; Divecha et
al., 1995). Although the function of these lipids has not yet been
determined, increasing evidence suggests that they may serve as
intracellular second messengers. PI 3-kinase is a heterodimer
consisting of a p85 regulatory subunit with SRC homology domains (SH2
and SH3) and a p110 catalytic subunit. A major mode of activation by
growth factors likely involves docking of PI 3-kinase through SH2
domains of the p85 subunit to phosphorylated tyrosine residues(s) of
tyrosine kinase receptors (Rordorf-Nikolic et al., 1994).
Moreover, activation of PI 3-kinase by growth factors may occur via
either Ras-dependent (Kodaki et al., 1994) or Ras-independent
(Rodriguez-Viciana et al., 1994) pathways. In either
situation, activation of Ras is sufficient to activate mitogenic
responses in a variety of cells. In other important cases, such as G
protein-coupled receptors, PI 3-kinase has been shown to be directly
activated by subunits released from activated G proteins
(Zhang et al., 1995).
In the present study, we have found
that adrenergic receptor-stimulated mitogenic
responses, such as DNA synthesis and activation of MAP kinase in
HVSMCs, are inhibited by wortmannin, a specific inhibitor of PI
3-kinase. This result suggests that activation of PI 3-kinase is
associated with
adrenergic receptor-stimulated
responses in HVSMCs. We further demonstrate directly that stimulation
of
receptors activates PI 3-kinase via a pertussis
toxin-sensitive G protein pathway. Moreover, noradrenaline-stimulated
PI 3-kinase is associated with activation of Ras proteins and tyrosine
protein kinases.
To characterize signaling pathways involved in receptor stimulation of mitogenic responses in vascular smooth
muscle cells, we tested the capacity of wortmannin, a specific
inhibitor of PI 3-kinase, to inhibit DNA synthesis. Wortmannin (10
nM) completely blocked noradrenaline-stimulated DNA synthesis
as well as platelet-derived growth factor-induced DNA synthesis (Fig. 1A). Noradrenaline's action was also
blocked by the
receptor-selective antagonist
terazosin. To determine whether the
receptor-mediated
increase in [
H]thymidine incorporation was
mediated via pertussis toxin-sensitive G proteins, cells were
preincubated with pertussis toxin (100 ng/ml) for 12 h before
stimulation with noradrenaline. Noradrenaline induced an 89% increase
of [
H]thymidine incorporation under control
conditions; preincubation with pertussis toxin markedly inhibited the
noradrenaline-induced increase in DNA synthesis in these cells (Fig. 1A). Since MAP kinase has been postulated to play
a key role in mediating mitogenic responses of many receptors,
including
adrenergic receptors in myocytes, we also
examined
adrenergic receptor-mediated activation of
MAP kinase in HVSMCs (Fig. 1B). Noradrenaline (1
µM) stimulated an approximately 2-fold increase in MAP
kinase activity in the presence of a
receptor antagonist timolol.
The
receptor antagonist terazosin (1 µM)
blocked noradrenaline-activated MAP kinase activity (Fig. 1B). Noradrenaline-stimulated activation of MAP
kinase was significantly attenuated by a 12-h preincubation of cells
with pertussis toxin (100 ng/ml) (about 30% increase over basal) and
partially blocked by inhibitor of PI 3-kinase wortmannin (about 43%
increase over basal) (Fig. 1B). Increased MAP kinase
activity was not inhibited by the protein kinase C inhibitor H7. These
results suggest that activation of PI 3-kinase as well as activation of
MAP kinases are involved in mediating catecholamine-induced mitogenesis
in human vascular smooth muscle cells. In addition, we found that IGF-I
increased MAP kinase activity in these cells and that this response was
inhibited by wortmannin but not by pertussis toxin; this result
suggests that pertussis toxin was not having nonspecific effects (Fig. 1B).
Figure 1:
adrenoreceptor
stimulation of DNA synthesis and activation of MAP kinase: effects of
inhibitors. A, HVSMCs purchased from Clonetics Corp. (San
Diego, CA) were grown to near confluence in a series of 35-mm
dishes. Quiescent cells were incubated with Dulbecco's
modified Eagle's medium containing 0.4% fetal calf serum plus
noradrenaline (1 µM) for 20 h in the presence of a
adrenoreceptor antagonist timolol (1 µM) (some cells had
been preincubated with 100 ng/ml pertussis toxin (PTx) for 12
h). Cells were then incubated with [
H]thymidine
(0.1 µCi) for another 4 h. Potential inhibitors were added to the
cell dishes 1 h before the addition of noradrenaline (Nor) as
indicated. Incorporation of [
H]thymidine into
cells was performed as described under ``Experimental
Procedures.'' The data are an average ± S.E. of three
experiments. B, the near confluent HVSMCs were incubated with
serum-free Dulbecco's modified Eagle's medium for 18 h.
Inhibitors were added into cell dishes 1 h before the addition of
agonists. After noradrenaline or vehicle treatment for 10 min, cells
were harvested for assay of MAP kinase activity as described under
``Experimental Procedures.'' The data are average ±
S.E. of three experiments. *, different from control, p <
0.05;**, different from control, p <
0.01
Several lines of evidence indicate that
PI 3-kinase plays an important role in growth regulation and
transformation. Analysis of mutations in the binding site for PI
3-kinase on the polyoma virus middle T antigen, which leads to either
failure to associate with PI 3-kinase or impaired capacity to elevate
the concentrations of PI 3-kinase products, have been found to result
in a transformation-defective phenotype (Fantl et al., 1992).
Similarly, point mutations in the PI 3-kinase binding sites of
platelet-derived growth factor receptors impair this receptor's
ability to stimulate DNA synthesis (Valius and Kazlauskas, 1993). Roche et al.(1994) have shown that microinjection of antibodies
specific for the p110 subunit of the PI 3-kinase into quiescent
fibroblasts inhibited platelet-derived growth factor-induced DNA
synthesis. Finally, a number of studies have demonstrated that
inhibition of PI 3-kinase by wortmannin, a specific PI 3-kinase
inhibitor, results in blockage of growth factors or serum-induced cell
proliferation (Panayotou and Waterfield, 1993; Varticovski et
al., 1994; Vemuri and Rittenhouse, 1994), inhibition of protein
kinase cascades (Ding et al., 1995), and suppression of growth
factor-induced blockade of apoptosis (Yao and Cooper, 1995).
Consequently, the present results demonstrate that wortmannin can block
noradrenaline-stimulated DNA synthesis and activation of MAP kinase,
suggesting that PI 3-kinase plays an important role in receptor-mediated mitogenesis in HVSMCs.
The capacity of a
whole range of tyrosine kinase receptors or TPKs (Cantley et
al., 1991; Rordorf-Nikolic et al., 1995; Van der Geer and
Hunter, 1991) to activate PI 3-kinase has been extensively studied; the
activated PI 3-kinase is believed to play an important role in signal
transduction pathways of peptide growth factors (Fry, 1994). On the
other hand, there is an increasing experimental evidence indicating
that PI 3-kinase may also be involved in signaling pathways of G
protein-coupling receptors; most of this evidence has been derived from
investigations of thrombin receptors in platelets (Stephens et
al., 1993; Zhang et al., 1995) and chemoattractant
receptors in neutrophils (Bokoch, 1995; Varticovski et al.,
1994). To determine directly if activation of receptors expressed in HVSMCs activated PI 3-kinase, we
implemented an in vitro assay of PI 3-kinase activity in
immunocomplexes precipitated by an antibody against the p85
subunit of PI 3-kinase (Yano et al., 1993). The results
demonstrated that noradrenaline stimulated a time-dependent activation
of PI 3-kinase at concentration of 10 µM in the presence
of a
adrenergic receptor antagonist timolol (1 µM) (Fig. 2A and Table 1). Noradrenaline stimulated a
very rapid and significant activation of PI 3-kinase that occurred as
early as 1 min after activation of
receptors, with
the peak activity at 5 min. The activity of PI 3-kinase declined to
basal values 30 min after continued exposure to noradrenaline.
Noradrenaline-stimulated activation of PI 3-kinase was also
concentration-dependent (Fig. 2B and Table 1). 1
µM of noradrenaline caused a significant increase in PI
3-kinase activity. However, the increased PI 3-kinase activity declined
at 100 µM of noradrenaline concentration, indicating that
blunted response of the
receptors to their ligand
occurred. Noradrenaline-activated PI 3-kinase was specifically mediated
by
receptors because the
-selective
antagonist terazosin inhibited this activation; on the other hand, the
adrenergic receptor antagonist timolol and
adrenergic receptor antagonist idazoxan did not inhibit
activation of PI 3-kinase (Fig. 3, lanes 2-4).
Additionally, activation of PI 3-kinase by noradrenaline was inhibited
by wortmannin (10 nM) as expected (Fig. 3, lane
6).
Figure 2:
Noradrenaline-stimulated time- and
concentrationdependent activation of PI 3-kinase. A, cells
were treated with vehicle or noradrenaline (10 µM) for the
indicated times. The whole cell lysates (1 mg of protein) were
subjected to immunoprecipitation with a rabbit polyclonal antibody
against the p85 subunit of PI 3-kinase. PI 3-kinase activity in
immunoprecipitates from noradrenaline-treated or control cells was
determined as described under ``Experimental Procedures.''
Change in activity of PI 3-kinase is presented as production of
phosphatidylinositol phosphate (PIP). The autoradiogram of TLC
of PI 3-kinase was exposed for 20 h. Experiments were repeated 3 times
with essentially identical results. B, cells were treated with
vehicle or the indicated concentrations of noradrenaline for 5 min, and
cell lysates were prepared and immunoprecipitated as described above.
The autoradiogram of TLC of PI 3-kinase was exposed for 24 h.
Experiments were repeated 3 times with essentially identical
results.
Figure 3:
Activation of PI 3-kinase inhibited by
adrenergic receptor antagonists and pertussis toxin.
Cells were pretreated with
receptor-selective
antagonist terazosin (10 µM) or the
receptor-selective antagonist idazoxan (10 µM), an
inhibitor of PI 3-kinase wortmannin (10 nM) for 2 h or with
pertussis toxin (PTx; 100 ng/ml) for 12 h. Cells were then
treated with vehicle, 10 µM of noradrenaline or 100 ng/ml
of IGF-I for 5 min. Cell lysates (1 mg of protein) were prepared,
immunoprecipitated with anti-p85
of PI 3-kinase antibody, and
subjected to determination of PI 3-kinase as described above. The
autoradiogram of TLC of PI 3-kinase was exposed for 24 h. Experiments
were repeated 3 times with similar results.
Thrombin and several chemoattractants stimulate cell
responses in several different cell types via activation of pertussis
toxin-sensitive G proteins leading to activation of PI 3-kinase. There
are several signaling pathways that are utilized by G protein coupling
to thrombin and chemoattractant receptors in the activation of PI
3-kinase (Bokoch, 1995; Stephens et al., 1993). One pathway
involves pertussis toxin-sensitive G proteins including P21
heterologous small G proteins such as Ras or Rho (Zhang et
al., 1995). Another pathway occurs via activation of the
traditional TPKs such as SRC kinases (Cantley et al., 1991).
For the latter pathway, activation of thrombin and chemoattractant
receptors leads to phosphorylation of cytosol TPKs, which in turn may
provide phosphorylated site(s) for binding of the p85 subunit of
PI 3-kinase through SH2 to TPKs. Additionally, a recent study has
identified a novel p110 isoform of the catalytic subunit of PI 3-kinase
that is activated without association with the p85 subunit of the
originally described PI 3-kinase heterodimer (Stoyanov et al.,
1995); interestingly,
subunits released from
receptor-activated G proteins directly activate this newly described PI
3-kinase catalytic moiety (Stephens et al., 1993). Although it
has been generally accepted that
receptor-stimulated
responses are likely predominantly mediated by
subunits released
by pertussis toxin-insensitive G proteins, likely in the Gq/11 family
(Schwinn et al., 1995), increasing evidence suggests that
pertussis toxin-sensitive G proteins may also be utilized to transduce
the signals of
receptor stimulation (Perez et
al., 1993). To test if pertussis toxin-sensitive G proteins are
involved in
receptor-mediated activation of PI
3-kinase, HVSMCs were preincubated with pertussis toxin (100 ng/ml for
12 h) and then stimulated by noradrenaline; as indicated in Fig. 3, lane 5, pertussis toxin completely blocked
activation of PI 3-kinase in these cells. However, IGF-I (100 ng/ml), a
well-known activator of a tyrosine kinase receptor, increased PI
3-kinase activity in these cells, but the response was not attenuated
by pertussis toxin (Fig. 3, lanes 7 and 8).
This result demonstrates the specificity of pertussis toxin in
inhibiting activation of PI 3-kinase by
receptors.
receptors are known to activate protein kinase C and
increase intracellular concentrations of Ca
. We found
that the L channel Ca
blocker nifedipine did not
inhibit noradrenaline-stimulated PI 3-kinase (data not shown);
4
-phorbol 12,13-dibutyrate, an activator of protein kinase C, did
not increase PI 3-kinase activity in these cells, suggesting that
activation of PI 3-kinase is independent of activation of protein
kinase C.
To determine if there was direct coupling of receptor to PI 3-kinase activity as has been found for tyrosine
kinase receptors,
or
receptors
were immunoprecipitated with specific antibodies directed against each
of these subtypes. The
or
receptor proteins could be recovered by these antibodies, but no
PI 3-kinase activity was detected in immunoprecipitates of
noradrenaline-treated cell lysates with either antibody, suggesting
that activation of PI 3-kinase by noradrenaline is transduced by
downstream mechanisms that do not invoke docking of PI 3-kinase with
these receptors (data not shown). To determine if Ras and TPKs are
involved in activation of PI 3-kinase by
receptors,
cell lysates from control or noradrenaline-treated HVSMCs were
immunoprecipitated with anti-Ras or anti-phosphotyrosine; PI 3-kinase
activity was measured in the immunoprecipitates (Fig. 4A). There was basal activity of PI 3-kinase
detected in Ras or tyrosine protein immunocomplexes from control cells;
however, a significant increase in PI 3-kinase activity could be
detected in both anti-Ras- or anti-phosphotyrosine immunocomplexes from
noradrenaline-stimulated cells (Fig. 4A and Table 2). These results demonstrate that both Ras protein and
tyrosine proteins are associated with
receptor-activated PI 3-kinase in HVSMCs. However, these data do
not provide the sequence of activation of PI 3-kinase, Ras protein, or
tyrosine kinases by
receptors. It is known that PI
3-kinase may act at either downstream (Kodaki et al., 1994) or
upstream of Ras protein (Rodriguez-Viciana et al., 1994) in
other cells. We further investigated the interaction of PI 3-kinase and
Ras protein in noradrenaline-stimulated HVSMCs; as illustrated in Fig. 4B, noradrenaline stimulated a time-dependent
increase in Ras-bound GTP in the presence of antagonists of
and
receptors, suggesting that activation of
receptors stimulates an increase in the active Ras-GTP. On
analysis of the temporal relationship between activation of PI 3-kinase (Fig. 2A) and increased active Ras protein (Fig. 4B), the active Ras-GTP appeared later than
activation of PI 3-kinase, suggesting that Ras protein might function
as a target of PI 3-kinase after stimulation of cells with
noradrenaline. This possibility was supported by the fact that
noradrenaline-stimulated increase in the active Ras-GTP could be
partially blocked by the specific inhibitor of PI 3-kinase wortmannin
as was terazosin, pertussis toxin, and genistein (Fig. 4C). We postulate that Ras protein is localized
downstream of PI 3-kinase and functions as a target of PI 3-kinase. The
definite interaction between the two important protein molecules in
HVSMCs by activation of
receptors will require
further investigation. Since increased activity of PI 3-kinase had been
found in anti-phosphotyrosine protein immunocomplexes, cells were
metabolically labeled with [
P]P
and
stimulated with or without noradrenaline. Cell lysates from control and
noradrenaline-treated cells were immunoprecipitated with antibodies
directed against the p85 subunit of PI 3-kinase and then resolved by
SDS-polyacrylamide gel electrophoresis. Noradrenaline stimulated
phosphorylation of the p85 subunit of PI 3-kinase (Fig. 4D, lanes 1-3). To determine if
noradrenaline stimulated tyrosine phosphorylation of PI 3-kinase, cell
lysates from control and noradrenaline-treated cells were
immunoprecipitated with anti-p85 antibody and then detected by
anti-phosphotyrosine antibody. Results indicated that noradrenaline
stimulated a tyrosine phosphorylation of p85 itself (Fig. 4E, lanes 1-3).
Figure 4:
Noradrenaline-activated PI 3-kinase
associates with activated Ras and a tyrosine-phosphorylated protein. A, cells were treated with vehicle or noradrenaline (10
µM) for 5 min. Cell lysates (500 µg of protein) were
subjected to immunoprecipitation (IP) with anti-H-Ras (lanes 1 and 2), anti-phosphotyrosine (anti-Tyr) (lanes 3 and 4), or anti-p85 of
PI 3-kinase (lanes 5 and 6). PI 3-kinase activity in
immunoprecipitates from noradrenaline-treated or control cells was
determined as described in Fig. 2. The autoradiogram of TLC of
PI 3-kinase was exposed for 16 h. Experiments were repeated twice with
essentially identical results. B, HVSMCs were metabolically
labeled with [P]P
for 12 h and
treated with noradrenaline (10 µM) in the presence of
timolol (1 µM) and idazoxan (1 µM) for the
indicated times. Cell lysates were prepared and immunoprecipitated with
anti-Ha-Ras antibody. TLC was used to separate GTP and GDP;
autoradiograms of TLC plates were exposed for 10 h. Ras-bound GTP
(percentage of GTP + GDP) was calculated with a PhosphorImager
system (Molecular Dynamics), and percentages are shown at the bottom. Experiments were repeated 3 times with essentially
identical results. C, HVSMCs were labeled as in B.
Inhibitors as indicated were added to dishes for 2 or 12 h (pertussis
toxin; PTX) before stimulation of cells with noradrenaline for
10 min. Changes in Ras-bound GTP were measured as in B. The
autoradiogram of TLC was exposed for 10 h. Experiments were repeated
twice with essentially identical results. D, HVSMCs were
metabolically labeled with [
P]P
for
12 h and treated with noradrenaline (10 µM) in the
presence of timolol (1 µM) and idazoxan (1
µM) for the indicated times. Cell lysates were prepared
and immunoprecipitated with anti-p85 of PI 3-kinase and then resolved
by SDS-polyacrylamide gel electrophoresis. The autoradiogram of the
film was exposed for 16 h. Experiments were repeated twice with
essentially identical results. This result indicates that noradrenaline
treatment stimulated a phosphorylation of the p85 of PI 3-kinase as
indicated by the arrow. E, cell lysates from control
or noradrenaline-treated cells were immunoprecipitated with anti-P85 of
PI 3-kinase and resolved by SDS-polyacrylamide gel electrophoresis.
Blots were probed with an anti-phosphotyrosine antibody. Experiments
were repeated twice with essentially identical results. This result
suggests that noradrenaline stimulates tyrosine phosphorylation of p85
as indicated by the arrow.
In summary, the
results demonstrate that receptors expressed in
HVSMCs are coupled to stimulation of PI 3-kinase via pertussis
toxin-sensitive G proteins. Activation of PI 3-kinase by noradrenaline
leads to association of the kinase with activation of Ras proteins and
TPKs. The results highlight the potential importance of
receptors in the activation of PI 3-kinase, particularly
concerning activation of mitogenesis in vascular smooth muscle cells.
Moreover, these results broaden concepts relating to interaction and
cross-talk of
adrenergic receptors with families of
tyrosine kinases.