(Received for publication, June 7, 1994; and in revised form, November 7, 1994)
From the
Angiotensin II (AII) is a growth factor which induces cellular
hypertrophy in cultured vascular smooth muscle cells (SMC). To
understand the molecular basis of this action, we have examined the
role of the 70-kDa S6 kinases (p70) in the hypertrophic
response to AII in aortic SMC. AII potently stimulated the
phosphotransferase activity of p70
, which reached a
maximal value at 15 min and persisted for at least 4 h. This response
was completely abolished when the cells were incubated in the presence
of the AT
-selective receptor antagonist losartan. The
enzymatic activation of p70
was associated with increased
phosphorylation of the enzyme on serine and threonine residues. The
immunosuppressant drug rapamycin was found to selectively inhibit the
activation of p70
by AII, but not the activation of
mitogen-activated protein kinase or the induction of c-fos mRNA expression. Treatment of aortic SMC with rapamycin also
potently inhibited AII-stimulated protein synthesis with a half-maximal
concentration similar to that required for inhibition of
p70
. These results provide strong evidence that
p70
plays a critical role in the signaling pathways by
which AII induces hypertrophy of vascular SMC.
Angiotensin II (AII) ()is a peptide hormone that
evokes a wide range of biological responses, including arteriolar
vasoconstriction, stimulation of aldosterone secretion, and renal
sodium reabsorption(1) . In addition, AII is a growth factor
for diverse cell types, such as fibroblasts, adrenocortical cells,
vascular SMC, and cardiac myocytes(2, 3) . In cultured
aortic SMC derived from normal rats, AII induces cell hypertrophy as a
result of increased protein synthesis, but not cell
proliferation(4, 5, 6, 7) . This
trophic response is associated with increased expression of mRNAs for
early growth response genes such as c-fos and c-myc(8, 9, 10) .
AII exerts its
physiological effects by interacting with two pharmacologically
distinct subtypes of receptors, designated AT and AT
(for review, see (11) and (12) ). Rat vascular
tissues express predominantly the AT
subtype, although the
rat aorta contains a small proportion of AT
receptors(13, 14) . Most of the major in
vitro and in vivo responses to AII are mediated by
AT
receptors (reviewed in Refs. 11, 12, and 15). The
AT
subtype is also responsible for the growth promoting
effects of AII in cultured cells (16, 17) . Both
AT
and AT
receptors belong to the superfamily
of seven transmembrane domain receptors, although G protein coupling
has not been demonstrated for the AT
receptor(18, 19, 20, 21) .
Activation of the AT
receptor triggers various G
protein-mediated signaling pathways, including stimulation of
phospholipases C, D, and A
and inhibition of adenylyl
cyclase(12) . Furthermore, in common with other G protein
agonists, AII stimulates tyrosine phosphorylation of multiple
substrates in target cells(22, 23, 24) . (
)However, the molecular basis of the hypertrophic response
to AII is still largely unknown.
A common response of cells to both
mitogenic and hypertrophic factors is the activation of protein
synthesis. This event is thought to be controlled in part by multiple
phosphorylation of the ribosomal protein
S6(25, 26, 27) . Phosphorylation of S6 at
five serine residues, all clustered at the carboxyl end of the protein,
is correlated with an activation of protein synthesis at the level of
initiation. Much effort has been directed toward identification of the
enzymes controlling S6 phosphorylation and elucidation of their mode of
regulation. Two distinct families of growth factor-regulated S6 kinases
have been characterized at the molecular level: the 90-kDa S6 kinase
family, referred to as p90(28, 29) ; and
the 70-kDa S6 kinase family, referred to as
p70
(30, 31) . Both classes of S6 kinases
are activated by serine/threonine phosphorylation, but their activities
are regulated by distinct signaling
pathways(27, 32, 33) . The p90
is phosphorylated and activated by the MAP kinase isoforms
p42
and p44
, which are themselves
regulated by complex protein kinase cascades (for recent reviews, see (34, 35, 36) ). The upstream components
responsible for p70
activation have not yet been
identified. While both the p90 and the p70 S6 kinases are able to
phosphorylate the 40 S ribosomal protein S6 in vitro, a number
of experimental results indicate that p70
is the major S6
kinase in vivo. Indeed, the p70
is highly
specific for S6, whereas p90
has wide substrate
specificity(33, 37) . Additionally, inhibition of
protein synthesis, which leads to S6 phosphorylation, is accompanied by
activation of p70
but not p90
(38) .
Finally, the immunosuppressant drug rapamycin, which blocks
serum-stimulated S6 phosphorylation, inhibits activation of p70
by mitogenic factors but has no effect on the activity of
p90
(39, 40, 41, 42) .
The aim of this study was to evaluate the involvement of p70 in the hypertrophic effect of AII on rat aortic SMC. We show that
AII stimulates the phosphorylation and enzymatic activation of
p70
in aortic SMC. In addition, we demonstrate that
p70
activity may play a critical role in the signaling
pathway leading to AII-stimulated protein synthesis.
The probes used were a 0.9-kilobase pair PstI fragment of mouse c-fos cDNA (provided by Dr. Mona Nemer, Montreal) labeled by random priming and a DNA oligonucleotide derived from the rat glyceraldehyde-3-phosphate dehydrogenase RNA sequence (provided by Dr. Louis Ferland, Montreal) 5`-end-labeled using T4 polynucleotide kinase.
Figure 1:
AII increases the rate
of protein synthesis in rat aortic SMC. Rat aortic SMC were made
quiescent by incubation in serum-free medium for 48 h. The cells were
then stimulated for 24 h with indicated concentrations of AII. Protein
synthesis was measured by [H]leucine
incorporation. Each value represents the mean of triplicate
determinations. A, dose-response curve for the stimulatory
effect of AII on protein synthesis. B, effect of AII receptor
antagonists. Quiescent aortic SMC were pretreated for 10 min with
medium alone or with the non-selective antagonist
[Sar
,Ile
]AII (sarile,
10
M), the AT
-selective
antagonist losartan (10
M), or the
AT
-selective antagonist PD123319 (3
10
M). The cells were then stimulated with medium or 100
nM AII for 24 h.
Figure 2:
AII stimulates the enzymatic activity of
p70 in rat aortic SMC. Quiescent rat aortic SMC were
treated for the indicated times with 100 nM AII. Cell lysates
were prepared and subjected to immunoprecipitation with p70
antiserum preadsorbed to protein A-Sepharose beads. Immune
complexes were washed, and p70
phosphotransferase
activity was assayed using an S6 peptide as substrate (see
``Experimental Procedures''). The enzymatic activities are
expressed as picomoles of PO
incorporated into the
substrate/min/mg of lysate protein. The data presented are
representative of three independent experiments with similar
results.
AII has been shown
previously to stimulate S6 phosphorylation in vascular SMC (55) and renal proximal tubular cells(56) . However,
the identity of the S6 kinases implicated has not been investigated.
Our present findings clearly established that AII potently stimulates
the enzymatic activity of the 70-kDa S6 kinase family in aortic SMC. In
this respect, we have also observed that AII activates the two MAP
kinase isoforms p44 and p42
in these
cells. However, the kinetic of activation of these enzymes is different
from the one of p70
. The activity of p44
and p42
increases rapidly to reach a maximum at 5
min and then declines rapidly to low value at 30 min. The activity of
the enzymes remains negligible thereafter. These observations are
consistent with the notion that MAP kinases and p70
lie
on distinct signaling pathways(57) .
To determine which
subtype of AII receptors is involved in the activation of
p70, rat aortic SMC were pretreated for 10 min with
selective receptor antagonists prior to stimulation with AII. Fig. 3shows that incubation with the AT
-selective
antagonist losartan completely abolished p70
activation,
whereas the AT
antagonist PD123319 had no effect. Thus,
these results demonstrate that stimulation of p70
activity, like most of the known biochemical responses to AII, is
mediated by AT
receptors.
Figure 3:
Effect of AII receptor antagonists on AII
stimulation of p70 activity. Quiescent rat aortic SMC
were pretreated for 10 min with medium alone or with the non-selective
AII receptor antagonist [Sar
,Ile
]AII (sarile, 10
M), the
AT
-selective antagonist losartan (10
M), or the AT
-selective antagonist PD123319
(3
10
M). The cells were then
stimulated with medium(-) or 100 nM AII for 15 min. The
p70
was immunoprecipitated from the cell lysates and S6
kinase activity was measured as described in Fig. 2. Similar
results were obtained in three independent
experiments.
Figure 4:
AII stimulates the phosphorylation of
p70 in rat aortic SMC. A, quiescent rat aortic
SMC were labeled with [
P]phosphoric acid for 5 h
and then stimulated or not (Cont) with 100 nM AII for
15 min. The cells were lysed, and p70
was
immunoprecipitated using a specific p70
antiserum
preadsorbed to protein A-Sepharose beads. The immunoprecipitated
proteins were resolved by SDS-gel electrophoresis on 10% acrylamide
gels and transferred to PVDF membranes prior to autoradiography.
Molecular weight standards are shown on the left. The
positions of p70
and the minor isoform p85
are indicated. B, quiescent rat aortic SMC were
stimulated or not (Cont) with 100 nM AII for 15 min.
Equal amounts of cell lysate protein (500 µg) were then subjected
to immunoprecipitation with either p70
antiserum (lanes1 and 2) or normal rabbit serum (lane3). The proteins were resolved on 7.5%
acrylamide gels and transferred to nitrocellulose membrane. The
membrane was probed with S6K-III antiserum and the proteins visualized
by chemiluminescence detection. The positions of p70
and
the minor p85
isoform are indicated. The p85
was hard to visualize on these immunoblots. Similar results were
obtained in two independent experiments.
To further analyze the changes in
the phosphorylation state of p70, the
P-labeled bands corresponding to p70
and to
the 85-kDa protein were subjected to phosphoamino acid analysis. In
quiescent aortic SMC, p70
was phosphorylated exclusively
on serine residues (Fig. 5). Stimulation of the cells with AII
resulted in a significant increase in the phosphoserine and to a much
lesser extent phosphothreonine content of p70
(Fig. 5). The increased phosphorylation of the 85-kDa
protein was also characterized by an augmentation of serine
phosphorylation, but the signal was too low to detect any change in
threonine phosphorylation (Fig. 5). Therefore, our results
demonstrate that activation of p70
by AII is associated
with increased phosphorylation of the enzyme mainly on serine but also
on threonine residues. It is noteworthy that stimulation of p70
by either epidermal growth factor in Swiss 3T3 cells (61) or insulin in H4 hepatoma cells (59) is associated
with a similar content of phosphorylated amino acids, thereby
suggesting that G protein-coupled receptors and receptor tyrosine
kinases might use the same upstream kinases to regulate the activity of
p70
.
Figure 5:
Phosphoamino acid analysis of
AII-stimulated p70. Quiescent rat aortic SMC were labeled
with [
P]phosphoric acid for 5 h and then
stimulated or not (Cont) with 100 nM AII for 15 min.
The p70
was immunoprecipitated from the cell lysates and
analyzed as described in Fig. 4. The
P-labeled
protein bands corresponding to p70
and the 85-kDa protein
were excised from the PVDF membrane and subjected to partial acid
hydrolysis. The phosphorylated amino acids were separated by
two-dimensional thin layer electrophoresis. The positions of
phosphoserine (S), phosphothreonine (T), and
phosphotyrosine (Y) are indicated.
We first tested the effects of rapamycin on p70 and MAP kinase activity in AII-stimulated aortic SMC. Consistent
with previous studies using different growth factors or cell types,
pretreatment of quiescent aortic SMC with 10 ng/ml rapamycin completely
abolished the activation of p70
by AII (Fig. 6A). Half-maximal inhibition was observed at a
concentration of 0.5 ng/ml rapamycin (n = 2) (Fig. 8). In contrast, rapamycin did not affect AII-induced
p44
activity as measured by the phosphorylation of MBP (Fig. 6B). To further demonstrate the selectivity of
rapamycin action, we evaluated the effect of the drug on induction of
the early-immediate gene c-fos in aortic SMC. AII stimulation
leads to increased c-fos mRNA expression in SMC and other cell
types. As shown in Fig. 7, AII-induced c-fos gene
induction was not inhibited by rapamycin. The normalized values of
c-fos induction, expressed as -fold stimulation above control,
were 10.1 in untreated cells stimulated with AII and 13.1 or 9.7 in
cells treated with 10 or 30 ng/ml rapamycin, respectively. These
findings also indicate that p70
activity is not required
for induction of c-fos gene expression. Similar observations
were made in an interleukin 2-dependent T-cell line where rapamycin was
found to block p70
activation by interleukin 2, but not
p90
activation, c-fos and c-myc mRNA
expression, or early tyrosine phosphorylation events(64) .
Taken together, these data indicate that rapamycin selectively inhibits
the activation of p70
among the early signaling events
triggered by AII in aortic SMC.
Figure 6:
Rapamycin selectively inhibits
AII-stimulated p70 activation in rat aortic SMC.
Quiescent rat aortic SMC were pretreated for 30 min with vehicle alone
or with the indicated concentrations (ng/ml) of rapamycin (rap). The cells were then stimulated or not (Cont)
with 100 nM AII for 15 min (p70
assays) or 5 min
(p44
assays). A, enzymatic activity of
p70
. The p70
was immunoprecipitated from
the cell lysates and S6 kinase activity was measured as described in Fig. 2. B, enzymatic activity of p44
.
The p44
was immunoprecipitated from the cell lysates
with antiserum SM1, and phosphotransferase activity was assayed using
MBP as substrate (see ``Experimental Procedures''). The
enzymatic activities are expressed as picomoles of PO
incorporated into the substrate/min/mg of lysate protein. The
data presented are representative of three independent experiments with
similar results.
Figure 8:
Rapamycin potently inhibits AII-stimulated
protein synthesis in rat aortic SMC. Quiescent rat aortic SMC were
pretreated for 30 min with the indicated concentrations of rapamycin
and then stimulated for 24 h with 100 nM AII in the continuous
presence of the inhibitor. Protein synthesis was measured by
[H]leucine incorporation (
). Each value
represents the mean of triplicate determinations. In the experiment
shown, the extent of stimulation of protein synthesis in response to
AII was 2.0-fold above basal level. The activity of p70 S6 kinase
(
) was determined as described in Fig. 6. Each point
represents the average from two independent experiments. AII stimulated
the S6 kinase activity of the enzyme 2.9-fold in the absence of
rapamycin. Similar results were obtained in four separate
experiments.
Figure 7:
Effect of rapamycin on AII-induced
c-fos gene expression. Quiescent rat aortic SMC were
pretreated for 30 min with vehicle or with the indicated concentrations
(ng/ml) of rapamycin (rap). The cells were then stimulated or
not (Cont) with 100 nM AII for 30 min. Total RNA was
extracted from the cells and analyzed by Northern hybridization using a P-labeled c-fos cDNA fragment as described under
``Experimental Procedures.'' The results were normalized by
rehybridization of the blots with a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) oligonucleotide probe. The extent of
hybridization was visualized by autoradiography and quantitated by
laser densitometry. Similar results were obtained in two independent
experiments.
We next examined the effect of
rapamycin on AII-stimulated protein synthesis in aortic SMC. For these
experiments, quiescent SMC were preincubated for 30 min with rapamycin
prior to stimulation with 100 nM AII for 24 h in the
continuous presence of the drug. Fig. 8shows that rapamycin
very significantly inhibited AII-induced protein synthesis with over
75% inhibition observed at 3 ng/ml of the drug. Half-maximal inhibition
of protein synthesis was observed in the presence of 0.3 ng/ml
rapamycin (n = 4), identical to the concentration
required for inhibition of p70 S6 kinase. The ED value
found for inhibition of AII-induced protein synthesis is comparable to
those observed for inhibition of interleukin 2-stimulated DNA synthesis
in T lymphoid cell lines(40, 64) . These findings
demonstrate that there is a close correlation between the stimulation
of p70
activity by AII and its hypertrophic effect in
SMC. It should be noted, however, that although rapamycin totally
abrogated AII-stimulated p70
activity, the drug never
completely inhibited (
60-80% inhibition) the increase in
protein synthesis. This suggests that additional signaling pathways are
recruited by AII to regulate the rate of protein synthesis. Future work
will be required to identify these other signaling pathways and
determine their relationship with the p70
pathway.
The present data provide strong evidence that activation of
the p70 signaling pathway by AII represents a critical
event in the hypertrophic response of SMC to the hormone. We showed
that AII potently stimulates the enzymatic activity of p70
in aortic SMC. This activation is mediated by AT
receptors and is associated with increased phosphorylation of the
enzyme on serine and threonine residues. The AII-induced p70
activation is completely blocked by rapamycin, whereas MAP kinase
activation or c-fos gene induction is not affected. Rapamycin
also potently inhibited AII-stimulated protein synthesis in these
cells. Considering the role of p70
as the major in
vivo S6 kinase, our findings strongly suggest that this enzyme
plays a critical role in the hypertrophic response of vascular SMC to
AII.
Another conclusion provided by these results is that
stimulation of MAP kinase activity and c-fos mRNA expression
are not sufficient to significantly increase the rate of protein
synthesis in SMC. Activation of MAP kinase isoforms is required for
cell cycle progression and S phase entry of fibroblast cells in
response to mitogenic factors(43, 65) . These enzymes
are rapidly translocated into the nucleus after mitogenic stimulation
of quiescent cells where they can phosphorylate and regulate the
activity of several transcription factors(66) . It has been
suggested that MAP kinases may also play a crucial role in the growth
promoting activity of AII(67, 68) . Our results do not
support the conclusion that MAP kinases alone are sufficient
intermediates in the signaling pathway between the AT receptor and the ribosomes. The exact role of the MAP kinase
signaling system in the response of vascular SMC to hypertrophic
hormones such as AII remains to be determined.