Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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In addition to the
well-documented role of nitric oxide (NO) as a vasodilator, NO has also
been implicated in vascular smooth muscle cell (VSMC) growth arrest.
Signaling mechanisms responsible for growth factor receptor-mediated
VSMC proliferation include the extracellular signal-regulated kinase
(ERK) and possibly the protein kinase B (PKB) cascade. Thus the present
study was designed to test the hypothesis that, in A7r5 vascular smooth
muscle-derived cells, platelet-derived growth factor (PDGF)-induced
activation of either ERK or PKB is regulated by NO, which then
modulates cellular proliferation and/or apoptosis. PKB- was the
predominant isoform of PKB expressed in A7r5 cells and was also
expressed in rabbit carotid arteries and aortae. Phosphorylation of
PKB-
and ERK induced by PDGF-BB was maximal within 5-15 min in
A7r5 cells. Preincubation of A7r5 cells with the NO donor
S-nitroso-N-acetylpenicillamine (SNAP) resulted
in a biphasic regulation of PDGF-stimulated PKB-
phosphorylation and
bioactivity. Acute exposure to SNAP significantly augmented
PDGF-induced activation of PKB-
, whereas prolonged incubation led to
a marked diminution in PDGF-induced activation of PKB-
. In contrast,
SNAP did not affect PDGF-induced activation of ERK at any time point.
The cGMP-independent effects of SNAP on PDGF-induced activation of
PKB-
were established with the use of an inhibitor of soluble
guanylyl cyclase, ODQ, as well as a cell-permeable analog of cGMP,
8-bromo-cGMP. Prolonged treatment of A7r5 cells with SNAP led to a
significant decrease in DNA synthesis without an appreciable increase
in apoptosis. These data suggest that, after prolonged exposure to
SNAP, NO selectively attenuates PDGF-induced increase in PKB-
activation, which in turn may contribute to diminished VSMC
proliferation by mechanisms involving growth arrest but not apoptosis.
protein kinase B; extracellular signal-regulated kinase; nitric oxide; growth factors; proliferation; platelet-derived growth factor; vascular smooth muscle cell
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INTRODUCTION |
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PLATELET-DERIVED GROWTH FACTOR (PDGF) is a mitogen responsible for vascular smooth muscle cell (VSMC) proliferation and is intimately involved in the development of vascular proliferative lesions observed in atherosclerosis and in restenosis after angioplasty (11, 32). In vivo administration of neutralizing antibodies to PDGF before and after balloon catheter deendothelialization results in a considerable decrease in neointimal smooth muscle accumulation (11). The endothelial layer in the vascular wall is known to exert a regulatory influence on vascular tone and VSMC proliferation under normal physiologic conditions, in part, by its ability to release nitric oxide (NO) upon activation of endothelial NO synthase (eNOS) (37). After balloon angioplasty, NO production via eNOS is shown to be diminished due to endothelial denudation (36), which may contribute to abnormal VSMC proliferation. In vivo transfer of the eNOS gene in denuded rat carotid arteries (37) and the administration of an NO donor (34) after balloon angioplasty result in suppression of abnormal VSMC proliferation, thus supporting the antiproliferative effects of NO. However, the cellular mechanisms governing the antiproliferative effects of NO are not completely understood (34, 37).
The role of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase cascade, in growth factor receptor-induced increases in DNA synthesis and proliferation of VSMC has been extensively studied (12, 17, 21). The signaling events following activation of PDGF receptors include sequential activations of ras GTPase, raf, MEK1 (mitogen-activated protein kinase kinase), and subsequently ERK1/ERK2. In addition to ERK activation, recent studies have demonstrated that PDGF-induced increases in DNA synthesis and proliferation of bovine tracheal smooth muscle cells may involve activation of additional kinase cascades linked ostensibly to cell survival, including phosphatidylinositol 4,5-bisphosphate 3-kinase (PI-3K) and protein kinase B (PKB) (38). Thus a novel signaling pathway involving activation of PKB may also play a pivotal role toward cell survival and proliferation in VSMC. However, as yet, there are no reports documenting which, if any, of the PKB isoforms are expressed or activated by growth factors in VSMC.
PKB (also called Akt) is a serine/threonine protein kinase that exists
as three distinct isoforms: PKB-, PKB-
, and PKB-
(4, 24, 25). PKB is clearly
established as one of the downstream targets of the lipid kinase PI-3K
(4, 13). Studies involving transient
expression of dominant-negative mutants of PI-3K, and also
pretreatments of cells with two chemically different PI-3K inhibitors,
LY-294002 and wortmannin, have confirmed the obligatory involvement of
PI-3K in the growth factor-induced activation of PKB (4,
13, 19). The known substrates for
phosphorylation by PKB include Bad and caspase-9, which when
phosphorylated prevent apoptotic cell death (4). A recent
study indicates that activation of PKB is intimately involved in the
activation and phosphorylation of eNOS, thereby facilitating NO
production (10, 15). However, there are no
reports documenting a putative negative-feedback regulatory effect of
NO on growth factor receptor-induced activation of PKB in any cell
type, including VSMC.
NO is known to stimulate cytosolic soluble guanylyl cyclase to increase cGMP formation with the accompanying activation of cGMP-dependent protein kinase (2, 5, 23, 31, 33). It has been shown that the NO donors, including S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside, exert an inhibitory effect on VSMC proliferation by cGMP-dependent (40) and cGMP-independent (2, 5, 20, 27) mechanisms. The putative role of NO/cGMP to modulate PKB-dependent mitogenesis has not yet been investigated.
The present study has examined PKB isoform protein expression in A7r5 vascular smooth muscle-derived cells and intact rabbit vascular tissues. In addition, the differential effects of the NO donor SNAP on PDGF-induced activation of both PKB and ERK have been studied before and after selective inhibition of soluble guanylyl cyclase with H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). To determine whether the inhibitory effects of SNAP on PDGF-induced activation of PKB lead to diminished proliferation or to induction of apoptosis, the alterations in DNA synthesis and the number of cells undergoing apoptotic cell death were monitored after prolonged exposure to SNAP.
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MATERIALS AND METHODS |
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Materials.
PDGF-BB, insulin-like growth factor I (IGF-I), and DMEM were purchased
from GIBCO (Grand Island, NY). Endothelin (ET)-1 was obtained from the
Peptide Institute (Tokyo, Japan). The antibodies to PKB-, PKB-
,
and phospho-ERK as well as the protein kinase A inhibitor peptide were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody
to PKB-
and the apoptosis detection kits were from Upstate
Biotechnology (Lake Placid, NY). (Ser-473)phospho-PKB-
antibody was
purchased from New England Biolabs (Beverly, MA). Histone H2B was from
Boehringer Mannheim (Indianapolis, IN). SNAP and
N-acetylpenicillamine were from Sigma Chemical (St. Louis, MO). ODQ and LY-294002 were from Calbiochem (San Diego, CA). The MEK1
inhibitor PD-98059 was from Biomol Research Laboratories (Plymouth
Meeting, PA). [
-32P]ATP (sp act 4,500 Ci/mmol) and
[3H]thymidine (sp act 6.7 Ci/mmol) were obtained from ICN
Pharmaceuticals (Costa Mesa, CA). All other chemicals were from either
Fisher Scientific (Fair Lawn, NJ) or Sigma Chemical (St. Louis, MO).
Cell culture and treatments. Rat embryonic thoracic aorta smooth muscle-derived A7r5 cells were obtained from American Type Culture Collection (Rockville, MD). The cells were incubated in DMEM, supplemented with 10% FBS at 37°C in a humidified atmosphere of 95% air-5% CO2. After attainment of confluency (~80%), the cells were incubated in DMEM devoid of serum for 20 h. The serum-starved cells were then exposed to PDGF-BB, IGF-I, ET-1, SNAP, LY-294002, PD-98059, ODQ, and 8-bromo-cGMP at the indicated concentrations and time intervals as described in the legends to the respective figures.
Preparation of tissue extracts from rabbit aortae and carotid arteries. New Zealand White rabbits weighing ~3 kg were used in this study. Animal living conditions are consistent with standards required by the Association for Assessment and Accreditation of Laboratory Animal Care International. The rabbits were killed by injecting Nembutal (100 mg/kg). The right carotid arteries as well the aortae were dissected free of adventitial fat and washed in ice-cold PBS. The tissues were then homogenized, using a polytron, under ice-cold conditions.
Western blot analyses.
The control and treated A7r5 cells were washed twice with ice-cold PBS
and exposed to lysis buffer [20 mM HEPES (pH 7.5), 40 mM NaCl, 50 mM
NaF, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 20 mM -glycerophosphate, 0.5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin] for 20 min. The cell lysates or the tissue homogenates were vortexed for
10 s and centrifuged at 14,000 g at 4°C for 10 min.
The supernatants (20 µg) from A7r5 cell lysates or from the
homogenates of rabbit aortae and carotid arteries were mixed with the
sample buffer [0.062 M Tris · HCl (pH 6.8), 2% SDS, 5%
-mercaptoethanol (vol/vol), 10% glycerol, and 0.01% bromphenol blue] and boiled for 5 min. The proteins were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Hybond-C, Amersham). The membranes were incubated overnight with the
blocking buffer consisting of 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.5). After washing the membranes with TBS (10 min × 3), the membranes were probed with appropriate primary antibodies (1 in 1,000 dilution) for 2 h at room temperature. After being washed
three times with TBS, the membranes were incubated in TBS containing
the appropriate secondary antibody (1 in 6,000 dilution) for 2 h
at room temperature. After three more washes with TBS, the protein
bands were detected by the enhanced chemiluminescence method and
quantified by laser densitometry. For some experiments, the membranes
were incubated in the stripping buffer [62.5 mM Tris (pH 6.7), 2%
SDS, and 100 mM
-mercaptoethanol] for 35 min at 55°C, blocked
with nonfat dry milk, and reprobed with a different primary antibody.
The efficiency of the stripping procedure was determined by excluding
the primary antibody and verifying for the absence of signals on the
stripped membrane. The protein concentration was determined by the
Bradford method.
Assay of PKB- activity.
The control and treated cells were washed twice with ice-cold PBS and
exposed to lysis buffer (pH 7.5) for 20 min. The cell lysates were
vortexed for 10 s and centrifuged at 14,000 g at 4°C
for 10 min, after which the supernatants (protein concentration 100 µg) were incubated overnight at 4°C with a primary antibody specific for the PKB-
isoform. Subsequently, IgG agarose was added
to the respective samples and incubated at 4°C for an additional 4-5 h. After centrifugation, the respective immunoprecipitates of
PKB-
were washed with the lysis buffer, followed by a wash with the
kinase buffer [50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM
MgCl2, 10 mM MnCl2, 10 mM
-glycerophosphate,
and 0.5 mM sodium orthovanadate]. The bioactivity of PKB-
was
determined (in vitro kinase assay) by incubating the immunoprecipitates
of PKB-
at room temperature for 20 min in 30 µl of kinase buffer
in the presence of 1 µM protein kinase A inhibitor peptide, 50 µM
unlabeled ATP, and 6 µCi [
-32P]ATP, using exogenous
histone H2B (1.5 µg/assay tube) as the substrate (26).
The reactions were terminated by adding 10 µl of 4× Laemmli sample
buffer, after which the samples were heated at 95°C for 5 min. The
proteins in the samples were resolved by 12% SDS-PAGE and transferred
to nitrocellulose membrane, after which autoradiography was performed
for determining the incorporation of 32P-labeled phosphate
into histone H2B. As a negative control, supernatants of lysates from
the control group of cells were subjected to mock immunoprecipitation
without the addition of anti-PKB-
antibody. These mock
immunoprecipitates did not reveal any bands in the in vitro kinase assay.
Measurement of nitrite. A7r5 cells were incubated in DMEM in the absence or presence of SNAP (100 µM) for 10 min or 18 h, after which media were collected and used for nitrite measurements. Nitrite levels in the media were quantitated colorimetrically by the Griess reaction according to the manufacturer's instructions (Calbiochem, San Diego, CA).
Determination of cGMP. Serum-starved A7r5 cells were preincubated in the absence or presence of ODQ (10 µM) at 37°C for 1 h. Subsequently, both untreated and ODQ-treated cells were exposed to SNAP (100 µM) for 10 min or 18 h. At the appropriate time points, media were aspirated, and the cells were washed twice with ice-cold PBS and extracted with acidified methanol (50% methanol and 0.1 N HCl). After lyophilizing these extracts, the samples were processed for cGMP measurements by RIA (cGMP 125I scintillation proximity assay system, Amersham). The protein was determined by the Bradford method.
[3H]thymidine incorporation. A7r5 cells were grown to confluency (~80%) in DMEM supplemented with 10% FBS, after which they were deprived of serum by incubation in DMEM devoid of FBS for 20 h. Subsequently, the cells were supplied with the fresh DMEM devoid of FBS and pretreated with the following agents: SNAP (100 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (10 µM, 2 h), and PD-98059 (50 µM, 2 h). Both control and treated cells were then exposed to PDGF-BB (10 ng/ml) or IGF-I (50 ng/ml) for an additional 20 h in the presence of the above agents. During the last 4-h incubation period, the cells were labeled with [3H]thymidine (0.5 µCi/ml). After labeling, the cells were washed three times with ice-cold PBS and then exposed to 10% trichloroacetic acid (TCA; 10 min × 3). After complete removal of the TCA, the acid-insoluble material was extracted with 0.1 N NaOH, and the incorporation of [3H]thymidine into DNA was determined with a liquid scintillation counter (6).
Cell counts. Serum-starved A7r5 cells were preincubated in the absence or presence of SNAP (50 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (1 µM, 2 h), or PD-98059 (5 µM, 2 h), after which the cells were stimulated with PDGF-BB (10 ng/ml) for 3 days. The cells were supplied with fresh serum-free DMEM containing the appropriate agents every 24 h. The cells were then collected using 0.06% trypsin and counted in a hemocytometer.
Measurement of apoptosis. A7r5 cells were grown on eight-well Lab-Tek slides (Nalge Nunc International). Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL) method (Upstate Biotechnology). Briefly, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Tween for 15 min, incubated with TdT end-labeling cocktail for 60 min, blocked for 20 min, and then incubated with avidin-FITC in the dark for 30 min. As a positive control, the permeabilized cells were incubated with DNase I for 60 min. To quantitate apoptotic cell death, the percentage of TUNEL-positive cells to total cells was calculated after counting the number of TUNEL-positive cells and total cells in five random fields, at a magnification of ×100, using a fluorescent microscope.
Statistical analyses. The results are expressed as means ± SE of 3 or more experiments. The data were analyzed by repeated-measures one-way ANOVA followed by Bonferroni t-test or Kruskal-Wallis one-way ANOVA followed by Dunn's method. The differences between means were considered statistically significant when P < 0.05.
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RESULTS |
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Detection of PKB isoform(s) in A7r5 cells, rabbit aortae, and
carotid arteries.
Initial experiments characterized PKB isoform(s) protein expression in
A7r5 cells (Fig. 1A).
Western blot analyses revealed a marked expression of PKB-
protein with more modest expressions of PKB-
and PKB-
proteins in
A7r5 cells. PC12 (pheochromocytoma) cells expressed all three isoforms
of PKB (1) and thus served as a positive control. To
confirm PKB-
protein expression in intact vascular tissues, the
homogenates of rabbit aortae and carotid arteries were probed with
anti-PKB-
antibody (Fig. 1B). In comparison with aortic
tissues, carotid arteries were characterized by relatively more
abundant PKB-
protein levels.
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Comparison of the stimulatory effects of PDGF-BB, IGF-I, and ET-1
on the phosphorylation of PKB- and ERK in A7r5 cells.
Although the ERK signaling cascade has been implicated in mitogenesis,
the role of PKB in cell proliferation is not clearly established.
Hence, we assessed PKB-
and ERK1/2 phosphorylations in A7r5 cells
after stimulation with the following mitogens: PDGF-BB or IGF-I, which
activate receptors possessing intrinsic tyrosine kinase activity, or
ET-1, which activates G protein-coupled receptors (Fig.
2, A-C).
Preliminary experiments revealed that maximal phosphorylations of
either PKB-
or ERK1/2, by PDGF-BB, IGF-I, or ET-1, occurred between
5 and 15 min. Hence, a time point of 5 min was chosen in this study.
The relative increases in PKB-
and ERK phosphorylations on
stimulation with PDGF-BB (10 ng/ml) were much higher in comparison with
that observed with the other two agonists, IGF-I (50 ng/ml) and ET-1
(100 nM). The increases in PKB-
phosphorylations in A7r5 cells after
stimulation with PDGF-BB, IGF-I, and ET-1 were ~163-fold, 24-fold,
and 5-fold, respectively, in comparison with the unstimulated cells
(n = 4), as measured after 5 min. Under similar assay
conditions, the increases in ERK2 phosphorylations after stimulation
with PDGF-BB, IGF-I, and ET-1 were ~34-fold, 4-fold, and 4-fold,
respectively, when compared with the unstimulated cells (n = 3). The phosphorylation profile for ERK1 in A7r5 cells after
stimulation with PDGF-BB, IGF-I, and ET-1 remained essentially the same
as that for ERK2.
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Effects of PI-3K inhibitor and MEK1 inhibitor on the basal
and PDGF-BB-induced phosphorylation of PKB- and ERK.
Figure 2, D and E, shows the effects of the
PI-3K inhibitor LY-294002 and the MEK1 inhibitor PD-98059 on
PDGF-BB-induced increases in phosphorylation of PKB-
and ERK2. After
preincubating A7r5 cells with 10 µM LY-294002 for 2 h, the
PDGF-BB-induced increase in PKB-
phosphorylation was completely
abolished (P < 0.05). Pretreatment of A7r5 cells with
50 µM PD-98059 for 2 h also led to a significant attenuation of
PDGF-induced increase in ERK2 phosphorylation (P < 0.05). LY-294002 and PD-98059 did not produce significant changes in
the basal phosphorylations of PKB-
and ERK2, respectively.
Time dependency for PDGF-BB-induced phosphorylation of PKB- and
ERK.
We next assessed PDGF-augmented PKB and ERK phosphorylation as a
function of time (Fig. 3A).
Maximal PDGF-induced PKB-
phosphorylation was observed at the time
points ranging between 5 and 15 min, followed by gradual decline to
basal levels. Similar time-dependent effects on ERK phosphorylation
were also observed on stimulation of A7r5 cells with PDGF (Fig.
3B).
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Differential effects of the NO donor SNAP on PDGF-BB-induced
phosphorylation of PKB- and ERK.
Because we hypothesized that exposure of VSMC to NO leads to growth
arrest, we next examined the effects of the NO donor SNAP on
PDGF-BB-induced increases in PKB-
and ERK2 phosphorylations in A7r5
cells (Fig. 4, A and
B). A biphasic response to PDGF-BB-stimulated increases in
PKB-
phosphorylations was observed on exposure of A7r5 cells to 100 µM SNAP. Acute pretreatments of A7r5 cells with SNAP for 10 and 30 min were found to cause a significant enhancement in PDGF-BB-stimulated
increases in PKB-
phosphorylations by 46 ± 16% (P < 0.05) and 85 ± 32% (P < 0.05), respectively, in
comparison with that observed in the control cells. Although a slight
increase in PDGF-BB-stimulated increase in PKB-
phosphorylation was
still observed upon pretreatment with SNAP for 2 h, this increase
was not statistically significant. When the preincubation time with SNAP was further extended to 6 h, there was an attenuation of PDGF-BB-stimulated increase in PKB-
phosphorylation by ~21%, which however is not statistically significant. Prolonged incubation with SNAP for 18 h resulted in a significant attenuation
(P < 0.05) of PDGF-BB-induced increase in PKB-
phosphorylation by 57 ± 16%. Similarly, a 24-h preincubation
with SNAP caused a significant attenuation (P < 0.05) of
PDGF-BB-induced increase in PKB-
phosphorylation by 63 ± 3%.
In contrast to PKB-
, pretreatment of A7r5 cells with 100 µM SNAP
did not significantly alter PDGF-BB-induced increases in the
phosphorylation of ERK2 at any of the time points shown in Fig. 4,
A and B. Although SNAP, by itself, produced a
marginal increase in the basal phosphorylation of ERK2 (Fig.
4A), statistical analyses of the data from several
experiments (n = 4) revealed that there were no significant
changes in the basal phosphorylations of ERK or PKB-
following
exposure to SNAP alone. As a control, pretreatment of A7r5 cells with
the inactive analog of SNAP, N-acetylpenicillamine (100 µM), for up to 18 h did not lead to any changes in
PDGF-BB-induced increases in PKB-
phosphorylation (n = 3). Furthermore, SNAP (100 µM)-induced release of NO was assessed by
measuring nitrite levels in the incubation medium. After exposure of
A7r5 cells to SNAP (100 µM) for 10 min and 18 h, nitrite levels
in the media were found to be 1.9 ± 0.14 and 25.2 ± 1.7 µM, respectively (n = 4). There were no detectable amounts
of nitrite in the media collected from the control group of cells.
These data suggest that NO released from SNAP is responsible for the
observed effects on PKB-
phosphorylation.
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Effects of SNAP on PDGF-BB-induced activation of PKB-, as
assessed by in vitro kinase assay.
The biphasic effects of SNAP on PDGF-BB-induced increases in PKB-
phosphorylations were verified by immunoprecipitating PKB-
from
unstimulated and PDGF-BB-stimulated cells after prior treatment with
SNAP and by observing the changes in the phosphorylation states of the
exogenous substrate histone H2B (Figs. 5,
A-D). These in vitro kinase assays utilizing
immunoprecipitated PKB-
showed that the PDGF-BB-induced increase in
PKB-
activity was subject to inhibition by LY-294002, thus
supporting the obligatory role of PI-3K in PDGF-BB-induced activation
of PKB (Fig. 5, A and B). It should be noted that
the increase in the activation of PKB-
by PDGF, as assessed by the
in vitro kinase assay, is less pronounced than that seen in
immunoblotting procedures using phosphospecific PKB-
antibodies.
Recently, Summers et al. (35) have shown that the in vitro
kinase assay, utilizing anti-PKB antibody, produces a much higher basal
kinase activity than that observed using anti-phospho-PKB antibody
(35). The observation of a relatively less pronounced
increase in PKB-
activity after stimulation with PDGF may be due to
an increase in the assay background in the present experimental
conditions. Nevertheless, confirming the Western blot experiments
utilizing phosphospecific antibodies, acute exposure of A7r5 cells to
100 µM SNAP for 10 min caused a significant augmentation of
PDGF-BB-stimulated increase in PKB-
activity (Fig. 5C).
In addition, prolonged treatment of A7r5 cells with 100 µM SNAP for
18 h led to a significant attenuation in PDGF-BB-stimulated
increase in PKB-
activity. Pretreatment of A7r5 cells with the
inactive analog of SNAP, N-acetylpenicillamine (100 µM),
for either 10 min or 18 h did not affect PDGF-BB-induced increases
in PKB-
activity (data not shown). Figure 5D shows that
exposure of A7r5 cells to increasing concentrations of SNAP (10 µM,
100 µM, and 1 mM) for 18 h resulted in a significant
dose-dependent attenuation in PDGF-BB-stimulated increases in PKB-
activities.
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Effects of ODQ and 8-bromo-cGMP on SNAP-induced changes in
PDGF-BB-stimulated phosphorylation of PKB-.
Pretreatment of A7r5 cells with a selective inhibitor of NO-sensitive
soluble guanylyl cyclase, ODQ, resulted in a complete inhibition of
SNAP-induced increase in cGMP levels (Table
1). Hence, experiments involving the use
of ODQ and 8-bromo-cGMP (a cell-permeable, nonhydrolyzable stable
analog of cGMP) were performed to determine whether cGMP mediates the
biphasic effects of SNAP on PDGF-BB-stimulated PKB-
phosphorylations. As shown in Fig. 6A, the attenuation of
PDGF-BB-stimulated increase in PKB-
phosphorylation, after exposure
to 100 µM SNAP for 18 h, was still observed in the presence of
10 µM ODQ. ODQ, by itself, did not affect PDGF-BB-stimulated increase
in PKB-
phosphorylation. The augmentation of PDGF-BB-stimulated increase in PKB-
phosphorylation, observed after exposure of A7r5
cells to 100 µM SNAP for 10 min, also remained unaffected by prior
treatment of these cells with 10 µM ODQ for 1 h (data not
shown). In addition, preincubation of A7r5 cells with 100 µM
8-bromo-cGMP for 1 h did not affect PDGF-BB-induced increase in
PKB-
phosphorylation. Figure 6B shows that
PDGF-BB-stimulated increase in ERK2 phosphorylation was not altered
significantly after prior treatments with SNAP, ODQ, or 8-bromo-cGMP.
These data suggest that both acute and chronic SNAP regulations of
PDGF-stimulated PKB-
activity are independent of cGMP.
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Effects of SNAP on PDGF-BB- or IGF-I-induced increase in
[3H]thymidine incorporation.
We next investigated if chronic SNAP treatment altered DNA synthesis as
assessed by [3H]thymidine incorporation into
acid-insoluble DNA. Figure 7A
shows that prolonged (21-22 h) treatments with 100 µM SNAP, 10 µM LY-294002, or 50 µM PD-98059 reduced both basal as well as
PDGF-BB-induced increases in [3H]thymidine incorporation.
ODQ did not reverse the inhibitory effects of SNAP upon PDGF-BB-induced
[3H]thymidine incorporation, thereby suggesting that
SNAP-induced increase in cGMP is not responsible for this inhibition.
The inhibitory effects of SNAP, independent of cGMP, were also observed
with another growth factor, IGF-I. These data demonstrate that SNAP could exert an inhibitory effect not only for PDGF-induced increase in
DNA synthesis, but also for IGF-I-induced increase in DNA synthesis.
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Effects of SNAP on PDGF-BB-induced increase in cell proliferation. Stimulation of A7r5 cells with PDGF (10 ng/ml) for 3 days led to an increase in cell number by 71 ± 18% (P < 0.05) in comparison with the control group of cells (Fig. 7B). The total number of cells in the control group was 32,250 ± 3,403 per well (n = 3). Pretreatment of cells with SNAP followed by stimulation with PDGF led to an increase in cell number by 11 ± 10%, which was not significantly different from control. Inclusion of ODQ and SNAP, followed by stimulation of cells with PDGF, resulted in an increase in cell number by 8 ± 22%. These data further demonstrate that the inhibitory effect of SNAP on PDGF-induced proliferation of A7r5 cells was not reversed by ODQ. Additionally, PDGF-induced increases in cell number after pretreatment with LY-294002 and PD-98059 were 1 ± 8 and 13 ± 12%, respectively.
Effects of SNAP on apoptotic cell death.
We next asked if the effects of SNAP or LY-294002 on proliferation
correlated with the induction of apoptosis. As shown in Fig.
8, exposure of A7r5 cells to 100 µM
SNAP for 18 h did not lead to appreciable apoptosis. The
percentage of TUNEL-positive cells after SNAP treatment was 3.25 ± 0.15% in comparison with a control value of 0.95 ± 0.15%.
When A7r5 cells were subjected to coincubation with 100 µM SNAP and
PDGF-BB (10 ng/ml), for 18 h, the percentage of TUNEL-positive
cells was 1.15 ± 0.05%. The exposure of A7r5 cells to the PI-3K
inhibitor LY-294002 (10 µM) in the absence and presence of PDGF-BB
(10 ng/ml) for a similar time interval produced significant apoptosis
to the extent of 34 ± 6 and 22 ± 3%, respectively. The
permeabilized cells incubated with DNase I served as positive control,
wherein nearly 96-98% of the cells were TUNEL positive. Trypan
blue exclusion studies showed that exposure of A7r5 cells to 100 µM
SNAP for 18 h did not induce cellular necrosis (data not shown).
The lack of apoptotic or necrotic damage suggests that NO released from
SNAP under these conditions promotes growth arrest without
cytotoxicity.
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DISCUSSION |
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NO released from endothelial cells may affect VSMC proliferation
under normal and diseased states. The present study using A7r5 cells
documents that exogenous NO diminishes PDGF-induced proliferation
without producing appreciable apoptosis. The antimitogenic actions of
NO are mediated through a novel mechanism involving regulation of
PKB-, but not ERK phosphorylation. The regulation of PKB-
by NO
appears to be independent of cGMP. These data implicate the PKB
signaling cascade as an important mitogenic pathway that is subject to
modulation by NO in VSMC.
PKB, which has earlier been shown to be expressed in different cell
types such as human umbilical vein endothelial cells (9) and rat fibroblasts (13), is also expressed in vascular
smooth muscle-derived A7r5 cells. The predominant isoform of PKB
expressed in A7r5 cells is PKB-, which is followed by PKB-
and
PKB-
. To know whether the A7r5 cell line would provide a model for
the vascular tissues in vivo, attempts were also made to determine the
relative expressions of PKB-
in two different intact vascular tissues from rabbits. PKB-
protein was detected in freshly isolated aortae as well as in carotid arteries from rabbits. The apparent difference in the expression levels of PKB-
between the aortae and
carotid arteries may simply be due to a difference in the cellularity
of the two vessels. This is the first report demonstrating the relative
protein expression levels of PKB isoform(s) in A7r5 cells, and of
PKB-
in intact vascular tissues. However, the preponderance of the
cell types in the intact vascular tissues that express distinct PKB
isoforms is not known at the present time. Further studies involving
histochemical techniques with the cryosections of vascular tissues
would delineate the relative distributions of PKB isoforms in the
vascular wall in normal and diseased states.
Previously, Graves et al. (17) demonstrated that PDGF-BB,
but not IGF-I, stimulates ERK activity in human newborn aortic VSMC. In
line with these studies, our data suggest that, in addition to ERK
activation, PKB- also undergoes preferential activation by PDGF-BB
in rat A7r5 cells. A transient maximal increase in the PDGF-induced
phosphorylation of both PKB-
and ERK2, within a time frame of
minutes, suggests that the phosphorylated forms of these kinases are
subject to rapid dephosphorylation by cell type-specific phosphatases.
However, in human umbilical vein endothelial cells, shear
stress-induced increases in PKB-
phosphorylation remain
elevated for up to 6 h (9).
There is little information on the potential biological agents that
facilitate or antagonize PKB activation in VSMC. The effects of the
vasodilator NO on VSMC proliferation have been extensively studied
(2, 5, 27, 34,
40). However, none of these previous studies has examined
the possible regulatory effects of NO on PKB activation in VSMC. The
present study shows that acute exposure of A7r5 cells to SNAP resulted
in marked augmentation of PDGF-induced increases in PKB-
phosphorylation and enzyme activity. In contrast, prolonged exposure of
A7r5 cells to SNAP led to marked diminutions in PDGF-induced increases
in PKB-
phosphorylation and activity. It is known that SNAP, which
has a half-life of ~5 h, releases NO spontaneously (20).
The accumulation of the stable oxidation product of NO, nitrite, in the
media, after exposure of A7r5 cells to SNAP for 18 h, was
~13-fold higher than that observed at the 10-min exposure time to
SNAP. Additionally, the inactive analog of SNAP,
N-acetylpenicillamine, which does not possess the
S-nitroso group, did not produce any alterations in the
basal or PDGF-induced activation of PKB-
for up to 18 h. These
data suggest that NO, released from SNAP, is responsible for the
observed dual effects under either acute or prolonged treatment
conditions. Further studies involving immunoprecipitation of PKB-
or
PKB-
, followed by in vitro kinase assay, would delineate the likely
activation of PKB-
and/or PKB-
by PDGF and the possible regulation of these activated isoforms by NO.
The biphasic/dual effects of NO donors have earlier been reported in
numerous cell types, including rat cardiomyocytes and human mesangial
cells (8, 23). NO donors, including SNAP, have been shown to initially augment and then diminish cyclooxygenase-2 expression in mesangial cells as a function of time (8).
In addition, Kojda et al. (23) have shown that low
concentrations of NO donors, including SNAP, stimulate cardiac
contractility, whereas high concentrations of SNAP cause a depression
of cardiac contractility. The physiological significance of enhanced
PDGF-induced PKB- activation after acute exposure to SNAP is not
known. Nevertheless, the diminished PDGF-induced PKB-
activation
after prolonged exposure to NO may adversely affect VSMC proliferation.
The mechanism responsible for the enhanced activation of PKB with PDGF on acute exposure to SNAP is unknown. A potential mechanism may involve activation of ras, because acute exposure of human Jurkat T cells to an NO donor, sodium nitroprusside, causes S-nitrosylation of the cysteine118 residue in ras (7, 28). This in turn leads to activation of PKB by a PI-3K-dependent, wortmannin-sensitive mechanism (7). However, in A7r5 cells, it appears that this enhanced activation by SNAP occurs downstream of ras for the following reasons. 1) SNAP has no effect on basal PKB phosphorylation, and 2) SNAP does not affect the ERK cascade, which is ras activated.
Studies were performed to investigate whether cGMP mediates the
diminution in PDGF-induced activation of PKB- after prolonged exposure (18 h) of A7r5 cells to SNAP. A selective inhibitor of NO-sensitive soluble guanylyl cyclase, ODQ, has previously been shown
to block SNAP-induced elevations in cGMP levels in rat aortic VSMC
(27), human umbilical vein endothelial cells
(18), and rat cardiomyocytes (33). Our data
show that ODQ also inhibits SNAP-induced increase in cGMP in A7r5
cells. Furthermore, the present experiments involving the use of ODQ
and 8-bromo-cGMP suggest that the attenuation of PDGF-induced
activation of PKB-
, following prolonged exposure to SNAP, occurs by
a mechanism independent of cGMP. It is possible that, on prolonged
exposure of A7r5 cells to SNAP, the NO released from SNAP may exert
inhibitory effects on PI-3K or directly on PKB by mechanisms involving
S-nitrosylation of cysteine residues and/or nitration of
tyrosine residues in these enzyme proteins. Irrespective of the
mechanisms involved, it appears that NO modulation of PDGF signaling
affects only the PKB and not the ERK pathway.
[3H]thymidine incorporation and cell proliferation
studies suggest that prolonged exposure of A7r5 cells to NO would
result in inhibition of PDGF-induced increase in DNA synthesis and
proliferation, respectively, and that these effects could occur
independent of cGMP. In support of these present observations, recent
studies utilizing ODQ have also demonstrated that several NO donors
including SNAP could exert an inhibitory effect on DNA synthesis and
proliferation in rat aortic VSMC (27) and human umbilical
vein endothelial cells (18) by mechanisms independent of
cGMP. Furthermore, it has been demonstrated by Ishida et al.
(20) that SNAP can induce the expression of
cyclin-dependent kinase (Cdk) inhibitor, p21 Cip1, which in turn can
inhibit the G1/S transition phase of VSMC proliferation by
inhibiting cdk2. The present observations of diminished activation of
PKB- on prolonged exposure to SNAP suggest that NO may exert a
negative regulatory effect on PKB signaling pathway which may, in part,
be accountable for increased induction of Cdk inhibitor, p21 Cip1,
eventually leading to diminished VSMC proliferation.
Previous studies have shown that PDGF is a competence growth factor,
whereas IGF-I, ET-1, and ANG II act as progression growth factors in
VSMC (3, 22, 30). It has also
been reported that the mitogenic effect of IGF-I is relatively less
than that produced by PDGF in human VSMC (30), whereas the
role of ET-1 on the mitogenic response in VSMC remains controversial
(39). Because, in the present study, IGF-I also stimulated
PKB- phosphorylation (although of a lesser magnitude in comparison
with PDGF), we verified that NO exerts an inhibitory effect on
IGF-I-induced mitogenesis in A7r5 cells. Similar to the data observed
with PDGF, SNAP also exerted an inhibitory effect on IGF-I-induced DNA
synthesis independent of cGMP. IGF-I-induced increases in PKB-
phosphorylation and DNA synthesis were relatively less compared with
responses produced by PDGF, thereby suggesting a possible correlation
between the extent of PKB-
activation and the extent of
proliferation. Thus the antagonistic effects of NO on growth
factor-induced activation of PKB-
may have potential implications
for VSMC proliferation.
We tested whether the antagonistic effect of SNAP on the proliferation of A7r5 cells is coincident with the induction of apoptosis. Previous studies have shown that exposure of VSMC to a high concentration of an NO donor, sodium nitroprusside (500 µM), for 48 h results in apoptosis (14). However, in the present study, exposure of A7r5 cells to 100 µM SNAP for 18 h did not lead to appreciable apoptosis. The present data are consistent with the reports of Ishida et al. (20) in that a similar concentration of SNAP did not produce apoptosis in human umbilical VSMC, as determined by flow cytometry. The fact that LY-294002, and not SNAP, could induce apoptosis is intriguing. This suggests the possibility that NO has the potential to protect against apoptosis even when PKB is inhibited. In this regard, NO has been shown to directly inhibit caspase-3 activation via S-nitrosylation (29, 36).
Taken together, our data suggest that prolonged exposure of A7r5 cells
to NO results in marked attenuations of PDGF-induced activation of
PKB-, which in turn diminishes cell proliferation by a mechanism
involving growth arrest and not apoptosis. Future studies with animal
models subjected to angioplasty may reveal the role of activated PKB
isoform(s) in the genesis of neoplasia. Treatment with NO-generating
agents under these conditions has the potential to ameliorate this
abnormal smooth muscle proliferation by exerting a chronic inhibitory
effect on the PKB signaling pathway.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-53715 and a grant from the W. W. Smith Charitable Trust (to M. Kester) as well as by a beginning grant-in-aid from the American Heart Association, Pennsylvania-Delaware Affiliate (to L. Sandirasegarane).
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: L. Sandirasegarane, Dept. of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033 (E-mail: lxs51{at}psu.edu).
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.
Received 15 July 1999; accepted in final form 24 January 2000.
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