(Received for publication, July 9, 1996, and in revised form, January 22, 1997)
From the Nitric oxide-generating vasodilators inhibit
vascular smooth muscle cell proliferation. To elucidate the mechanism
underlying this process, we investigated the effect of
S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent,
on the smooth muscle cell cycle. When G0 cells were
stimulated with fetal bovine serum and basic fibroblast growth factor,
DNA synthesis assessed by [3H]thymidine incorporation
started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 µM. This inhibition was attenuated when SNAP was added
after 9-12 h. SNAP inhibited the activity of
cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the
retinoblastoma protein, both of which usually increased from about
9 h, whereas it did not inhibit the activities of cyclin
D-associated kinase(s), Cdk4, and Cdk6, which normally increased from
0-3 h. Although SNAP reduced the mRNA levels of cyclins E and
A, it neither reduced their protein levels nor impaired their
association with Cdk2. SNAP did not reduce the mRNA levels of
cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally
elevated from 0-3 h. The mRNA and protein levels of the Cdk
inhibitor p21 were high in the early G1 phase, peaking at
3 h and then rapidly decreasing after 6 h. In the presence of
SNAP, however, p21 expression was enhanced, and moreover, the later
decrease disappeared. SNAP also increased the amount of Cdk2-associated
p21. These results suggested that nitric oxide inhibits the
G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the
Cdk2 inhibition.
The proliferation of vascular smooth muscle cells
(VSMCs)1 plays a crucial role in the
formation of vascular lesions, such as fibrous plaques in
atherosclerosis and intimal thickening after balloon angioplasty (1,
2). The increase in smooth muscle mass in hypertensive vascular walls
may be related to an increase in cell number and DNA content (3).
Therefore, to understand the etiology of these disorders and to develop
new therapeutic strategies, it is essential to clarify the molecular
mechanism controlling VSMC proliferation.
The endothelium is the source of a variety of substances that control
vascular functions, such as nitric oxide (NO). NO inhibits platelet
adhesion and aggregation, leukocyte adhesion, and smooth muscle
contraction (4) and may also regulate VSMC proliferation. Vasodilators
that release NO, such as sodium nitroprusside, isosorbide dinitrate,
and S-nitroso-N-acetylpenicillamine (SNAP), inhibit the proliferation
of cultured VSMCs, probably by releasing NO (5). NO may also inhibit
VSMC proliferation in vivo because intimal thickening after
balloon angioplasty is prevented by L-arginine, the
metabolic precursor of NO (6), and by transferring plasmids that
express endothelial constitutive NO synthase into the vascular wall
(7). However, little is known about the signal transduction involved in
the antiproliferative effect of NO, although the involvement of cGMP
has been investigated (5, 8-11).
The eukaryotic cell cycle is regulated by cyclins and
cyclin-dependent kinases (Cdks). In mammalian cells, cyclin
D, cyclin E, and possibly cyclin A play important roles in controlling
the transition from G1 to S phase (12, 13). These cyclins
activate their catalytic partners Cdk4, Cdk6, and Cdk2 to
hyperphosphorylate the retinoblastoma protein (pRb) in the late
G1 phase (14), which then triggers entry into S phase. Cdk
activities are also controlled by the Cdk inhibitor proteins p21, p27,
p16, and p15 (13, 15).
Several extra- and intracellular signals inhibit cell proliferation.
Among them, transforming growth factor- SNAP was purchased from Research Biochemicals
International (Natick, MA) dissolved in Me2SO4,
and stored at VSMCs obtained from the media of human
umbilical arteries by explant cultures were used at the third passage.
Their origin was confirmed by immunostaining with anti-smooth muscle
To measure the DNA content, the cells were
dispersed with trypsin, suspended in phosphate-buffered saline, and
fixed with 85% (v/v) ethanol ( DNA synthesis was assessed by the level of
thymidine (TdR) incorporation. Cells seeded in 24-well plates (1 × 104 cells/well) were cultured in growth medium for
48 h. They were washed three times with serum-free Dulbecco's
modified Eagle's medium containing 0.1% bovine serum albumin and then
incubated in the same medium for 48 h for synchronization in
G0. The cells were labeled with 37 kBq/ml
[6-3H]TdR (0.74-1.1 TBq/mmol, Amersham Corp.) in growth
medium for the indicated periods. The medium was discarded and the
cells were washed three times with 0.5 ml of ice-cold
phosphate-buffered saline containing 1 mM MgCl2
and 1 mM CaCl2. The cells were precipitated with 0.5 ml of 5% trichloroacetic acid for 10 min, and then the acid
was removed with 0.5 ml of ethanol:diethyl ether (3:1, v/v). The
precipitates were lysed with 0.5 ml of 0.3 M NaOH,
neutralized with 2 M HCl, and mixed with 5 ml of
scintillation fluid (Aquasol-2, DuPont NEN Research Products).
Radioactivity was determined using a liquid scintillation counter
(LS5801, Beckman Instruments).
To prepare pRb
immunoprecipitates, about 3 × 106 cells were lysed in
1 ml of buffer A (10 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, and 0.5% (v/v) Nonidet P-40),
followed by centrifugation at 16,000 × g to remove the
insoluble pellet. The supernatant was incubated with 50 µl of 50%
(v/v) suspension of protein G-conjugated Sepharose (Pharmacia Biotech
Inc.) for 30 min on ice, followed by centrifugation to remove the
beads. The supernatant was incubated with an anti-human pRb monoclonal antibody (2 µg/ml, XZ104, PharMingen) for 1 h on ice.
Thereafter, 50 µl of a 20% (v/v) suspension of protein G-Sepharose
was added, and the incubation was continued for 1 h.
Immunoprecipitates of cyclin A, cyclin E, Cdk2, p21, p27, and p53 were
obtained in the same manner except for the use of polyclonal antibodies
to human cyclin A (Santa Cruz Biotechnology), cyclin E (Upstate
Biotechnology), Cdk2 (Upstate Biotechnology), p21 (Santa Cruz
Biotechnology), and p27 (Santa Cruz Biotechnology), and a monoclonal
antibody to human p53 (G59-12, PharMingen). Cyclin D and Cdk4
immunoprecipitates were prepared using polyclonal antibodies to human
cyclin D (Upstate Biotechnology) and Cdk4 (PharMingen), respectively,
after cells were lysed by sonication for 10 s in buffer B (50 mM Hepes/NaOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 0.1 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.1%
Tween 20, and 10% glycerol).
After five washes with buffer A or B, the precipitates were
fractionated by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a polyvinylidene difluoride membrane, and immunoblotted with the indicated antibodies. Second antibodies (horseradish peroxidase-linked anti-mouse or anti-rabbit
immunoglobulins) were visualized using the ECL Western blotting
detection system (Amersham Corp.).
Protein concentrations were measured by the modified Lowry's method
using a protein assay kit (Bio-Rad).
To measure the
activity of Cdk2, histone H1 and a pRb fragment were used as the
substrates. The Cdk2 immunoprecipitate was suspended in 40 µl of 20 mM Tris/HCl (pH 7.4) containing 10 mM MgCl2, 1 mM dithiothreitol, 50 µM
[ To assay the activities of cyclin D-associated kinase(s), Cdk4, and
Cdk6, GST-Rb (amino acid residues 754-921) prepared as described (29)
was the substrate. The immunoprecipitates of cyclin D, Cdk4, and Cdk6
were suspended in 40 µl of 50 mM Hepes/NaOH (pH 7.5)
containing 10 mM MgCl2, 1 mM
dithiothreitol, 20 µg/ml GST-Rb, and 50 µM
[ The cDNAs for human Cdc2, Cdk2, Cdk4,
Cdk5, and cyclins C, D1, E, and G were produced as described (26). The
cDNA for p21 was produced by means of the reverse
transcription-polymerase chain reaction, using RNA extracted from human
VSMCs as described (27) and a pair of oligonucleotides synthesized
according to the EMBL data base, and then it was subcloned into
pBluescript II SK(+) (Stratagene). The clone was sequenced to confirm
the identity of the product. The cDNAs for human cyclins A and B
were gifts from Jonathon Pines.
Equal amounts of total cellular RNA (10 µg/lane) were resolved by
electrophoresis on a 1% agarose gel containing 20 mM
3-(N-morpholino)propanesulfonic acid (pH 7.0), 5 mM
sodium acetate, 0.5 mM EDTA, and 6% formaldehyde. After
staining with ethidium bromide, RNA was transferred to a nylon membrane
and fixed by means of ultraviolet irradiation. The membranes were
incubated with cDNA probes labeled with 32P by
random-prime DNA labeling in 50% formamide, 10% polyethylene glycol,
7% SDS, 1 mM EDTA, 0.25 M NaCl, 0.25 M NaHPO4 (pH 7.2), and 100 µg/ml denatured
salmon sperm DNA for 16 h at 50 °C. They were washed with 15 mM NaCl containing 1.5 mM sodium citrate and 0.1% SDS at 65 °C and analyzed with a BAS-2000 bioimage
analyzer.
The results are expressed as the means ± standard deviation (S.D.) of the number of observations. The
statistical significance was assessed by Student's t test
for paired or unpaired values.
First we
determined whether or not SNAP inhibits cell proliferation. In growth
medium, the cell number increased exponentially until they reached
confluence around day 7 (Fig. 1). SNAP (100 µM) markedly inhibited the increase, and removing SNAP
from the medium recovered the proliferation. We confirmed by trypan
blue exclusion that the antiproliferative effect of SNAP did not result from cytotoxicity (data not shown). Flow cytometry showed that hypodiploid cells did not accumulate in the presence of 100 µM SNAP, indicating that this concentration did not
induce apoptosis (data not shown).
To examine whether the G1 and S phases are influenced by
SNAP, we synchronized cells in G0 phase by serum starvation
and stimulated them with growth medium. DNA synthesis, assessed by
measuring [3H]TdR incorporation, started about 15 h
after stimulation and reached a plateau at about 33 h (Fig.
2a). When SNAP (100 µM) was
added at 0-6 h, over 90% of the DNA synthesis was inhibited, but this
was attenuated when added later. These findings suggested that SNAP
inhibited the cell cycle in the late G1 phase. However, SNAP inhibited 15-20% of the incorporation when added after 18 h, indicating that SNAP also inhibited S phase to some extent. SNAP
dose-dependently inhibited the incorporation to levels of over 90% at concentrations above 100 µM (Fig.
2b). We then examined the effect of cPTIO, an NO scavenger
(30), on [3H]TdR incorporation, to test whether the
antiproliferative effect of SNAP is mediated by NO released from the
compound (Fig. 2c). Because a high concentration of cPTIO
and long exposure to cPTIO were cytotoxic, we pulse-labeled
unsynchronized cells with [3H]TdR in the presence of a
relatively low concentration (1-10 µM) of this compound.
Although cPTIO added alone reduced [3H]TdR incorporation,
it prevented the SNAP-induced inhibition of the incorporation.
To determine how it
inhibits DNA synthesis, we examined the effect of SNAP on pRb
phosphorylation because this is essential for cells to enter S phase
(14). We precipitated pRb with an antibody that recognizes both the
underphosphorylated and hyperphosphorylated forms and then
fractionated the precipitates by SDS-PAGE (Fig. 3a). The faster- and slower-migrating bands
(110 and 114 kDa) are hypophosphorylated and hyperphosphorylated
pRb, respectively. In G0 cells, the faster-migrating band
was dominant. The mobility shift of pRb from 110 to 114 kDa began at
about 9 h after stimulation and appeared to have been completed by
about 18 h. SNAP (100 µM) inhibited this shift,
suggesting that it blocked a G1 event that preceded pRb
phosphorylation. Like DNA synthesis, pRb phosphorylation was inhibited
dose-dependently on SNAP (Fig. 3b). To exclude
the possibility that pRb immunoprecipitates were unevenly loaded into the lanes and the SNAP-induced reduction of the hyperphosphorylated pRb
resulted from a diminished loading,
To elucidate the
mechanism by which SNAP inhibits pRb phosphorylation, we measured the
activities of Cdks because pRb is the putative substrate for Cdk2,
Cdk4, and Cdk6, which are activated during G1 phase (14).
Fig. 4a shows the effect of SNAP on Cdk2 activity measured using histone H1 and GST-Rb as the substrates. The
activity of histone H1 kinase was low in the G0 and early G1 phases. It began to increase about 9 h after
stimulation and continued to increase until 24 h. SNAP (100 µM) added at time 0 markedly suppressed the activities
measured at 12 and 24 h. SNAP also inhibited the GST-Rb kinase
activity of Cdk2, the time course of which was parallel to that of
histone H1 kinase activity.
Cyclin D and its major catalytic partners Cdk4 and Cdk6 may play
important roles in pRb phosphorylation (12-14). To examine the effects
of SNAP on the activities of cyclin D-associated kinase(s), Cdk4, and
Cdk6, we measured the kinase activity of their immunoprecipitates using
GST-Rb as the substrate. The activity of cyclin D-associated kinase(s)
was detectable in G0 cells, and it increased during early
G1 (3-7.5 h after stimulation). After it dropped during late G1 (at 9 h), the activity again increased from 12 until 24 h. SNAP did not affect the activation during early
G1, although the increase after the entry into S phase (24 h) was inhibited (Fig. 4b). Correspondingly, SNAP did not
inhibit the activities of Cdk4 and Cdk6 in the G1 phase
(Fig. 4c).
We examined the effects of SNAP on the mRNA expression
of cyclins by Northern blotting because association with a cyclin is required to activate Cdks (Fig. 5a). Cyclin G
was maximally expressed in the G0 and early G1
phases. Cyclins C and D1 were expressed at low levels in quiescent
cells, but they were induced during early G1. SNAP (100 µM) did not reduce the expression of any of these three
cyclins at least for the first 12 h but rather tended to enhance
their expression during G1.
It was difficult to define when the expression of cyclin E began (Fig.
5a). Small amounts of the mRNA were detected in early G1, and the expression was enhanced from 12-15 h. The
level of cyclin A mRNA was low for 12 h, and then it increased
from about 15 h, which was around the G1/S border.
Cyclin B mRNA levels appeared to begin increasing a few hours later
than cyclin A, probably after entry into S phase. In the presence of
SNAP, however, the levels of these three mRNAs were suppressed,
particularly after advance into S phase.
Fig. 5b shows Cdk gene expression. The level of Cdk2
mRNA was elevated during early G1 and further increased
after advancing into S phase. Cdk4 was markedly expressed even in
quiescent cells, and the level was not dramatically altered throughout
the G1 and S phases. Cdk5 expression was enhanced during
the G1 phase, but rather attenuated after 12 h. Cdc2
mRNA was not clearly detected until 24 h, suggesting that it
was expressed after entry into S phase. SNAP (100 µM)
suppressed the expression of Cdc2, which seemed to result from
inhibition of the G1/S transition. On the other hand, the
expression of Cdk2, Cdk4, and Cdk5 was not inhibited at least for the
first 12 h, indicating that SNAP did not inhibit their expression
during the G1 phase.
Because the mRNA levels of cyclins E and A were reduced
in the presence of SNAP, we tested by means of Western blotting whether SNAP also reduces their protein levels. The Western blots showed that
cyclins E and A were already present in quiescent cells (Fig. 6a). Mitogenic stimulation elevated the level
of cyclin A, but did not alter that of cyclin E. SNAP did not reduce
these levels, even at 24 h, at which time it obviously reduced
those of the corresponding mRNAs. Because SNAP did not alter the
protein level of Cdk2, we examined by immunoblotting the Cdk2
immunoprecipitates whether it inhibits the association of Cdk2 with
these cyclins (Fig. 6b). However, there was no significant
change in the levels of cyclins coprecipitated with Cdk2 in cells
exposed or not to SNAP.
SNAP did not reduce the protein levels of cyclin D and Cdk4, in
parallel with the results of the Northern blotting (Fig.
6c). SNAP did not reduce the amount of Cdk4 coprecipitated
with cyclin D, either, indicating that it did not inhibit the
association of Cdk4 with cyclin D.
Because
p21 inhibits the activity of cyclin E-Cdk2, cyclin A-Cdk2, cyclin
D1-Cdk4, and cyclin D2-Cdk4 complexes (31, 32), we investigated the
effect of SNAP on its expression. We detected p21 mRNA in quiescent
cells (Fig. 7a). The expression did not change, or rather increased during the first 3 h after stimulation but decreased after 6 h to levels that were much lower than those of quiescent cells. SNAP enhanced the expression at 3 h and,
moreover, prevented the decrease in the later phase. As a result, high
levels of expression continued up to 30 h in cells exposed to
SNAP. Western blotting showed a considerable amount of p21 protein in
G0 cells (Fig. 7b). The expression increased
3 h after stimulation but decreased thereafter. In the presence of
SNAP, however, there was little decrease in the amount of the protein,
resulting in continuous and elevated expression throughout the
observation period. To determine whether p21 induced by SNAP is
responsible for the inhibition of Cdk2 activity, we examined the effect
of SNAP on the level of p21 associating with Cdk2 (Fig. 7c).
The level of p21 coprecipitated with Cdk2 gradually increased in
response to growth stimulation. Although SNAP did not significantly
alter this level during the early G1 phase (3-6 h), it was
elevated in the presence of SNAP from the late G1 phase
(12-24 h).
On the other hand, the protein expression of p27, a Cdk inhibitor
related to p21 (33), was not altered during the G1 and S
phases, and there was no significant difference in its levels between
cells exposed to SNAP and those not exposed (data not shown).
p21 is transcriptionally up-regulated by the tumor suppressor gene
product p53 and is suggested to be involved in the p53-mediated growth
arrest in response to stimuli that damage DNA, such as ultraviolet
irradiation. To test whether SNAP induces p21 through the induction of
p53, we examined the effect of SNAP on the level of p53. The expression
of p53 increased after growth stimulation, but was not enhanced by SNAP
(Fig. 7d).
The effects of SNAP in this study were likely to be mediated by NO
released from the agent. First, SNAP spontaneously releases NO with a
half-life of about 5 h when added to neutral media exposed to room
air at 37 °C (34). Second, sodium nitroprusside, another NO-generating agent, also inhibited VSMC proliferation (data not shown). Third, the NO scavenger, cPTIO, attenuated the inhibitory effect of SNAP on DNA synthesis. High concentrations of NO may be
cytotoxic or could induce apoptosis. Several reports have suggested that NO induces apoptosis in macrophages (35, 36). Recent evidence
shows that NO released from VSMCs stimulated with interleukin-1 causes
them to induce their own apoptosis by up-regulating Fas and that
apoptosis is also induced in VSMCs by incubating them with 500 µM sodium nitroprusside for 48 h (37). However, SNAP up to 100 µM did not induce cell death as assessed by
trypan blue exclusion, and cells resumed proliferating after removing
SNAP. Flow cytometry showed that SNAP did not accumulate hypodiploid cells, indicating that the cells were not apoptotic. Therefore, we
assumed that the effects of SNAP were mediated by NO and that concentrations up to 100 µM did not induce cytotoxicity
or apoptosis.
The antiproliferative effect of NO-generating agents is not limited to
VSMCs. They also inhibit the proliferation of some other mammalian
cells, such as fibroblasts, renal mesangial cells, and venous
endothelial cells (9, 38, 39). This suggests that NO interferes with
cellular events that are common among some cell types. SNAP inhibited
the cell cycle of VSMCs in the G1 phase and, probably, also
those in the S phase. This is because its inhibitory effect on DNA
synthesis was attenuated when added in the late G1 phase,
but some effect remained when added after the advance into S phase.
Some mechanisms have been proposed for the S phase inhibition by NO. NO
gas and NO generated by activated macrophages inhibit ribonucleotide
reductase, a rate-limiting enzyme in DNA synthesis (40, 41). SNAP (over
100 µM) inhibits thymidine kinase, another enzyme
important for DNA synthesis, in VSMCs (42). However, the mechanism
underlying NO-induced G1 inhibition is unknown.
Several afferent signals that control cell cycle converge onto pRb
(14). Hypophosphorylated pRb binds to and inhibits several proteins
involved in initiating DNA replication, such as E2F transcription factors. The pRb hyperphosphorylated by Cdks during late G1
loses this ability and allows the cell to advance into S phase. Most of
the antiproliferative signals so far studied inhibit pRb
phosphorylation by reducing the expression levels and the activities of
cyclin-Cdk complexes responsible for pRb phosphorylation. Therefore, we
examined the effects of SNAP on pRb, cyclins, and Cdks.
SNAP inhibited late G1 events, namely Cdk2 activation and
pRb phosphorylation. Because Cdk2 phosphorylates pRb in
vitro (43) and may be a mediator of G1/S transition
(44), the inhibition of Cdk2 activity may explain the SNAP-induced
inhibition of pRb phosphorylation and the G1/S transition.
On the other hand, SNAP did not inhibit the cellular events that
started in early G1, such as the expression of cyclins G,
C, and D1, Cdk2, Cdk4, and Cdk5, or the activation of cyclin
D-associated kinase(s), Cdk4, and Cdk6. Cdk4 and Cdk6 may also be
involved in pRb phosphorylation (14). According to a recent report
(45), pRb has multiple Cdk consensus phosphorylation sites and each may
have a specific function for regulating pRb binding to various
proteins, such as SV40 large T-antigen, cAbl, and E2F. This implies
that Cdk4 and Cdk6 phosphorylate specific sites distinct from those
phosphorylated by Cdk2. If phosphorylation by Cdk2 is essential for pRb
inactivation, the SNAP-induced inhibition of Cdk2 would cause
G1 arrest despite the presence of active Cdk4 and Cdk6.
Associating with cyclin E or cyclin A is prerequisite for Cdk2
activation (12, 13). Because SNAP reduced the mRNA levels of
cyclins E and A, we initially considered that this suppressed the Cdk2
activity. However, SNAP did not reduce the protein levels of these
cyclins. Because the cyclin proteins were detected in quiescent cells,
they may undergo little degradation and their levels may be maintained
even if low levels of mRNA are expressed during the early
G1 phase. Therefore, we concluded that other mechanisms are
involved in the SNAP-induced inhibition of Cdk2 activity.
Cdk activities are negatively regulated by Cdk inhibitor proteins. p21,
also known as senescent cell-derived inhibitor 1 (Sdi1) (46),
Cdk-interacting protein 1 (Cip1) (31), and wild-type p53-activated
fragment 1 (Waf1) (47), binds to cyclin-Cdk2 complexes to inhibit their
activities (32). The overexpression of p21 inhibits pRb phosphorylation
and the proliferation of mammalian cells, including VSMCs (32, 48).
In vivo transfection with an adenovirus encoding p21
inhibits neointimal hyperplasia after balloon angioplasty of the rat
carotid artery (48). Apart from injurious stimuli, such as ultraviolet
irradiation, however, only a few physiological substances are known to
induce p21. These include transforming growth factor- We found that SNAP induces p21. p21 protein was abundant during early
G1, but its level decreased from about 9 h. A similar time course of p21 expression during G1 and S phases has
been identified in IMR90 fibroblasts (49). In the presence of SNAP, however, p21 expression was enhanced and did not decline. On the other
hand, Cdk2 activity was low up to 6 h after mitogenic stimulation, after which the level increased from about 9 h. SNAP suppressed this activation. This mirror image between p21 expression and Cdk2
activity suggested that p21 controls Cdk2 activity during the
G1/S progression. However, it is unclear whether p21
contributes to the suppression of Cdk2 activity during the early
G1 phase because the level of p21 associated with Cdk2 was
relatively low during the early G1 phase. SNAP elevated the
level of Cdk2-associated p21 from the late G1 phase,
suggesting that p21 is involved in the SNAP-induced Cdk2
inhibition.
SNAP did not influence the activities of cyclin D-associated kinase(s),
Cdk4, and Cdk6, although p21 reportedly inhibits the activity of cyclin
D-Cdk4 complex (31, 32). However, a recent report showed that
recombinant p21 nearly completely inhibits Cdk2 activity but only
partially affects Cdk4 and has no effect on Cdc2, suggesting that
sensitivity to p21 is different among different Cdks (50). Therefore,
p21 induced by SNAP may inhibit Cdk2 much more effectively than Cdk4
and possibly Cdk6. Although the precise mechanism for p21-induced Cdk2
inhibition is unknown, p21 may prevent phosphorylation of Cdk2 on
threonine-160 by Cdk-activating kinase (51). Additional study is needed
to determine whether SNAP inhibits the phosphorylation of Cdk2.
In addition to its ability to inhibit Cdks, p21 binds to the cofactor
of DNA polymerase p21 is induced by p53 and is thought to be important for cell cycle
arrest following DNA damage. However, recent studies indicate that p21
is up-regulated through p53-independent mechanisms in several
situations, including during normal tissue development, during cell
differentiation, following serum stimulation, and following treatments
with transforming growth factor- cGMP produced by soluble guanylate cyclase mediates NO-induced
vasorelaxation (57). NO-releasing agents stimulate cGMP formation, and
exogenous cGMP analogues mimic their effects on Ca2+
mobilization and smooth muscle tone (58). cGMP may also be involved in
the NO-induced inhibition of VSMC proliferation (5, 8, 10). The
cGMP-dependent activation of cAMP-dependent
protein kinase is partly responsible for the NO-mediated inhibition of VSMC proliferation (11). Nevertheless, NO-generating vasodilators inhibit the proliferation of BALB/c 3T3 fibroblasts that lack soluble
guanylate cyclase (9). In our VSMCs, high concentrations of
8-bromo-cGMP slightly inhibited [3H]TdR incorporation
(data not shown). We examined whether cGMP mediates the effect of SNAP
using inhibitors of cyclic nucleotide-dependent protein
kinases. The effect of SNAP was not reversed by Rp-GMPS or Rp-AMPS,
cGMP and cAMP analogues that inhibit cGMP- and
cAMP-dependent kinases, respectively (data not shown).
Therefore, whether or not cGMP is involved in the antiproliferative
effect of NO remains inconclusive.
VSMC hyperplasia is involved in the development of atherosclerotic
lesions, restenosis after balloon angioplasty, and vascular wall
thickening in hypertension (1-3). VSMCs in normal arteries are
abundant in contractile proteins and cannot proliferate (contractile type). However, the VSMCs in the neointima formed during
atherosclerosis and restenosis after angioplasty have de-differentiated
properties (59). Endothelial dysfunction may trigger a phenotypic
change in the VSMCs and induce their abnormal proliferation. This
implies that normal endothelium releases substances that differentiate VSMCs and inhibit their proliferation. The putative substances involved
in this role include NO, as well as other factors produced by the
endothelium, such as prostacyclin, adrenomedullin, C-type natriuretic
peptide, transforming growth factor- Here, we proposed a biochemical background for the NO-induced
inhibition of VSMC proliferation. However, there remain several unresolved issues, particularly the role of NO in VSMC differentiation. Because the cells we used here had the synthetic phenotype, we did not
study the effect of NO on reciprocal changes between contractile and
synthetic phenotypes. Recent reports suggest that p21 is involved in
the differentiation of skeletal muscle cells and macrophages (61, 62).
Therefore, it would be of interest to examine whether or not NO is
involved in regulating VSMC differentiation through p21 induction.
We thank Koshiro Fukiyama for giving A. I.
an opportunity to work with T. S., C. K., and J. O. in the National
Cardiovascular Center Research Institute. We are also indebted to
Jonathon Pines for providing the cDNAs of cyclins A and B and
Hitoshi Matsushime for the plasmids encoding GST-Rb.
Department of Bioscience,
Research Institute for Microbial Diseases,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(16-22), cAMP (23, 24),
protein kinase C (25, 26), and prostaglandin A2 (27, 28)
have been studied relatively in depth. Most of them inhibit
G1/S transition by preventing pRb phosphorylation, which
may result from reduced Cdk activities. To elucidate the mechanism
underlying the NO-induced inhibition of VSMC proliferation, we examined
the effects of SNAP on cellular events during the G1 and S
phases, including pRb phosphorylation, Cdk activation, and the
expression of cyclins, Cdks, and Cdk inhibitors, using VSMCs from human
umbilical arteries.
Chemicals
20 °C until use. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) was obtained from Dojindo Laboratories (Kumamoto, Japan). 8-Bromoguanosine 3
,5
-cyclic monophosphate (8-bromo-cGMP) was from
Sigma. Rp-8-bromoguanosine-3
,5
-monophosphorothioate
(Rp-GMPS) and Rp-8-bromoadenosine-3
,5
-monophosphorothioate (Rp-AMPS)
were from BioLog Life Sciences Institute (Bremen, Federal Republic of
Germany). Other common chemicals were of reagent grade.
-actin (Dako Corp.). The cells were maintained in Dulbecco's
modified Eagle's medium containing 20% (v/v) fetal bovine serum
(HyClone), 10 ng/ml human recombinant basic fibroblast growth factor
(Amersham Corp.), 100 units/ml penicillin, 100 µg/ml streptomycin,
and 1 µg/ml amphotericin B (growth medium). The cells were cultured in Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin in the absence of fetal bovine serum and basic fibroblast growth factor
for 48 h to synchronize them in a quiescent state
(G0). Thereafter, they were stimulated with growth medium
to reenter the cell cycle. Cell numbers were determined using a Coulter
counter (Z1).
20 °C) for 30 min. The ethanol was
removed and the cells were incubated in phosphate-buffered saline
containing RNase (172 Kunitz units/ml, Sigma) for 30 min at 37 °C, and then stained for 30 min with 0.005% propidium
iodide dissolved in phosphate-buffered saline. The fluorescence of the
DNA was measured using a flow cytometer (CytoACE150, Japan
Spectroscopic).
-32P]ATP (3.7 MBq/ml, Amersham Corp.), and 100 µg/ml histone H1 (Boehringer Mannheim) or 20 µg/ml glutathione
S-transferase-fused murine pRb carboxyl-terminal (GST-Rb)
(amino acid residues 769-921, Santa Cruz Biotechnology), and then
incubated for 30 min at 30 °C with occasional mixing. The reaction
was terminated with an equal volume of 2 × loading buffer (2%
SDS, 10% 2-mercaptoethanol, 125 mM Tris/HCl, pH 6.8, 20%
glycerol, and 0.04% bromphenol blue). The sample was boiled for 3 min,
and then the beads were precipitated by centrifugation. The supernatant
was fractionated by SDS-PAGE and transferred to a membrane.
Phosphorylated proteins were visualized using a bioimage analyzer
(BAS-2000, Fuji Photo Film Co.).
-32P]ATP (3.7 MBq/ml), and the mixtures were
incubated for 40 min at 30 °C with occasional mixing. The following
procedures were as described for the Cdk2 assay.
SNAP Reversibly Inhibited VSMC Proliferation
Fig. 1.
The effect of SNAP on VSMC
proliferation. VSMCs seeded in 6-well plates (3 × 104/well) were cultured in growth medium (). The medium
was changed every other day and the cells were counted every day.
Two-thirds of the cultures (
) were exposed to SNAP (100 µM) added every other day simultaneously with the
changing medium (filled arrows). On day 5 (open
arrow), one-half of the cultures treated with SNAP were placed in
SNAP-free growth medium after several washes (
), and the other half
were continuously exposed to SNAP. Data represent the means ± S.D. (n = 3). *, p < 0.01 versus control.
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
The effect of SNAP on DNA synthesis.
a, cells seeded in 24-well plates (1 × 104
cells/well) were cultured in growth medium for 48 h and then in
serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine
serum albumin for 48 h to synchronize the cells in G0
phase. The cells were stimulated with growth medium to reenter the cell cycle in the presence of [3H]TdR (37 kBq/well), and the
amount of accumulated radioactivity was measured at the indicated times
(). Closed circles (
) represent the SNAP-induced
inhibition (%) of [3H]TdR incorporation measured at
33 h against the values obtained with solvent alone (0.1%
Me2SO4). SNAP (100 µM) or vehicle
was added at the indicated times. Data represent the means ± S.D. (n = 3). b, G0 cells were
labeled with [3H]TdR in growth medium in the presence of
various concentrations of SNAP added at time 0, and then
[3H]TdR incorporation was measured at 33 h. Data
represent the means ± S.D. (n = 4). *,
p < 0.01 versus the value obtained in the
absence of SNAP. c, Cells seeded in 24-well plates (1 × 104/well) were cultured in growth medium for 48 h
and then labeled with [3H]TdR (37 kBq/well) for 4 h
in the presence or absence of various concentrations of cPTIO and 100 µM SNAP. Data represent the means ± S.D.
(n = 3). *, p < 0.05 versus
the value obtained in the absence of cPTIO.
[View Larger Version of this Image (20K GIF file)]
-tubulin was immunoblotted as a
protein loading control. As shown in Fig. 3c, there was no difference in the amount of
-tubulin between control and
SNAP-treated cells, whereas in the same samples, SNAP down-regulated
the hyperphosphorylated and up-regulated the hypophosphorylated
pRb. In addition, SNAP had no significant effect on the total protein
level (5.78 ± 0.34 µg/ml/104 cells in control and
6.40 ± 0.34 µg/ml/104 cells in SNAP-treated
cells).
Fig. 3.
The effect of SNAP on pRb phosphorylation.
a, subconfluent G0 cells were stimulated with
growth medium in the presence or absence of SNAP (100 µM)
and lysed at the indicated times. Immunoprecipitates prepared with the
antibody to pRb were resolved by SDS-7.5% PAGE and immunoblotted with
another monoclonal antibody to pRb (1.5 µg/ml, G3-245, PharMingen).
The arrows indicate hypophosphorylated (110 kDa) and
hyperphosphorylated (114 kDa) forms of pRb (pRb and
pRbphos., respectively). b,
G0 cells were stimulated with growth medium in the presence
or absence of various concentrations of SNAP. Cell lysates prepared at
24 h were analyzed as described in a. NS and
S, not stimulated and stimulated with growth medium,
respectively. c, pRb was blotted as described in
b in the absence or presence of SNAP (100 µM).
Equal proportions of the supernatant that remained after pRb
immunoprecipitation were fractionated by SDS-10% PAGE, transferred to
a membrane, and immunoblotted with a monoclonal antibody to -tubulin
(5 µg/ml, DM1A, Oncogene Research Products).
[View Larger Version of this Image (43K GIF file)]
Fig. 4.
The effects of SNAP on Cdk activities.
a, G0 cells were stimulated with growth medium in the
absence or presence of SNAP (100 µM). Cell lysates
prepared at the indicated times were immunoprecipitated with the
anti-Cdk2 antibody. After the kinase reaction using histone H1 or
GST-Rb as the substrate, the assay mixture was fractionated by SDS-PAGE
(acrylamide concentration was 12.5% for histone H1 and 10% for
GST-Rb), electroblotted onto a membrane, and visualized by
autoradiography. b, cell lysates prepared at the indicated
times were immunoprecipitated with the anti-cyclin D antibody. After
the kinase reaction using GST-Rb as the substrate, the assay mixture
was resolved by SDS-10% PAGE and analyzed as described in
a. c, the procedure was the same as described in
b except that anti-Cdk4 and anti-Cdk6 antibodies were used
for immunoprecipitation.
[View Larger Version of this Image (34K GIF file)]
Fig. 5.
The effects of SNAP on the mRNA
expression of cyclins and Cdks. a, G0 cells were
stimulated with growth medium in the presence or absence of SNAP (100 µM), and then total RNA was extracted at the indicated
times. Equal amounts of RNA (10 µg/lane) were resolved by
electrophoresis and hybridized with 32P-labeled cyclin
cDNA fragments. The sizes of the transcripts (in kilobases) were:
cyclin G, 2.7; cyclin C, 2.4; cyclin D1, 4.5; cyclin E, 1.9; cyclin A,
2.9; cyclin B, 1.9. b, Northern blotting proceeded as
described in a, using Cdk cDNA fragments. The sizes of
the transcripts (in kilobases) were: Cdk2, 2.5; Cdk4, 1.7; Cdk5, 1.4;
Cdc2, 1.6; -actin, 2.1.
[View Larger Version of this Image (85K GIF file)]
Fig. 6.
The effects of SNAP on the protein expression
of cyclins and Cdks. a, G0 cells were stimulated
with growth medium in the presence or absence of SNAP (100 µM). Cells were harvested at the indicated times, and
cell lysates were immunoprecipitated with the polyclonal antibodies to
cyclin E or cyclin A. Proteins were fractionated by SDS-10% PAGE and
immunoblotted with monoclonal antibodies to cyclin E (1.5 µg/ml,
HE12, PharMingen) or cyclin A (1.5 µg/ml, C160, PharMingen).
b, cell lysates extracted 24 h after growth stimulation
were immunoprecipitated with the anti-Cdk2 antibody, fractionated as
described in a, and immunoblotted with the same antibody to
Cdk2 or the monoclonal antibodies to cyclin E or cyclin A, as
indicated. c, cells stimulated as described in a
were harvested at 7.5 h, and then cell lysates were
immunoprecipitated with the antibodies to cyclin D or Cdk4. Proteins
were fractionated as described in a and immunoblotted with
the same antibodies to cyclin D or Cdk4 (1.5 µg/ml), as
indicated.
[View Larger Version of this Image (52K GIF file)]
Fig. 7.
The effect of SNAP on the expression of p21
and p53. a, G0 cells were stimulated with growth
medium in the presence or absence of SNAP (100 µM), and
total RNA was extracted at the indicated times. Equal amounts of RNA
(10 µg/lane) were resolved by electrophoresis and hybridized with
32P-labeled p21 cDNA fragment. b,
G0 cells were stimulated as described in a.
Cells were harvested at the indicated times, and then lysates were
immunoprecipitated with the antibody to p21. Proteins were resolved by
SDS-12.5% PAGE and immunoblotted with a monoclonal antibody to p21
(1.5 µg/ml, 6B6, PharMingen). c, procedures were the same
as described in b except that lysates were
immunoprecipitated with the antibody to Cdk2. d, cell
lysates prepared as described in b were immunoprecipitated
with a monoclonal antibody to p53 (1.5 µg/ml, G59-12, PharMingen).
Proteins were fractionated by SDS-10% PAGE and then immunoblotted with
the same antibody.
[View Larger Version of this Image (34K GIF file)]
(21, 22) and
prostaglandin A2 (28).
, proliferating cell nuclear antigen, and inhibits
proliferating cell nuclear antigen-dependent DNA replication (52, 53). These two distinct inhibitory activities of p21
reside in separate domains of the protein (54, 55). The amino- and
carboxyl-terminal domains of p21 interact with and inhibit Cdk2 and
proliferating cell nuclear antigen, respectively. Therefore, not only
the G1 inhibition but also the SNAP-induced inhibition of S
phase may involve p21, although other mechanisms have been proposed for
the NO-induced S phase inhibition, as described above.
and prostaglandin A2
(56, 22, 28). SNAP did not elevate the level of p53 protein. This
suggested that the growth arrest by SNAP is not secondary to DNA damage
and that p21 induction by SNAP does not depend on p53. Further
investigation is needed to determine whether SNAP stimulates the
transcription of p21 independently of p53 or stabilizes the
mRNA.
, and heparan sulfate
proteoglycans. The role of NO is evidenced by the observation that
nitro-L-arginine reduces the antiproliferative effect of endothelial cells on cocultured VSMCs (60) and that intimal thickening
after balloon angioplasty is prevented by L-arginine and NO
synthase (6, 7).
*
This study was supported in part by grants from the Ministry
of Health and Welfare (Research Grants for Cardiovascular Diseases 6B-1
and 8A-1), the Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology (Encouragement System of
Center of Excellence)), and the Japan Cardiovascular Research Foundation.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. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-6-833-5012; Fax: 81-6-872-7485; E-mail:
sasaguri{at}ri.ncvc.go.jp.
1
The abbreviations used are: VSMC, vascular
smooth muscle cell; NO, nitric oxide; SNAP,
S-nitroso-N-acetylpenicillamine; Cdk, cyclin-dependent
kinase; pRb, retinoblastoma protein; cPTIO,
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide;
8-bromo-cGMP, 8-bromoguanosine 3,5
-cyclic monophosphate; Rp-GMPS, Rp-8-bromoguanosine-3
,5
-monophosphorothioate; Rp-AMPS, Rp-8-bromoadenosine-3
,5
-monophosphorothioate; TdR, thymidine; PAGE,
polyacrylamide gel electrophoresis; S.D., standard deviation; GST-Rb,
glutathione S-transferase-fused murine pRb
carboxyl-terminal.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.