Vol. 277, Issue 2, H515-H523, August 1999
Failure of cdc2 promoter activation and
G2/M transition by ANG II and
AVP in vascular smooth muscle cells
Nobuya
Fujita1,
Yusuke
Furukawa2,
Naoki
Itabashi1,
Yasushi
Tsuboi1,
Michio
Matsuda2,
Koji
Okada1, and
Toshikazu
Saito1
1 Division of Endocrinology and
Metabolism, Department of Medicine, and
2 Division of Molecular
Hemopoiesis, Center for Molecular Medicine, Jichi Medical School,
Tochigi 329-0498, Japan
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ABSTRACT |
The physiological
role of the vasoconstrictive hormones arginine vasopressin (AVP) and
angiotensin II (ANG II) in the development of vascular hyperplasia is
still unclear. We examined the effects of these hormones on cell cycle
regulation of cultured rat vascular smooth muscle cells (VSMC). AVP and
ANG II were able to induce G1/S
transition and DNA synthesis in serum-starved quiescent VSMC but failed
to promote further progression into
G2/M phases. AVP and ANG II
enhanced the expression and activity of cdk2, cyclin E, and
proliferating cell nuclear antigen but did not induce expression of
cdc2/cyclin B complex, a critical regulator of
G2/M transition. The failure of
cdc2 mRNA induction was found to be caused by a defect in cdc2 promoter
activation. Binding of free E2F-1 to the cdc2 promoter did not occur in
hormone-treated VSMC, which may account for the defective induction of
cdc2. The absence of cdc2 promoter activation and
G2/M transition may be important
for the prevention of hyperplasia under physiological conditions but
underlies the hypertrophy of VSMC.
cell cycle; proliferating cell nuclear antigen; cyclin; E2F-1
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INTRODUCTION |
HYPERPLASIA of vascular smooth muscle cells (VSMC) is a
fundamental pathogenic feature of hypertension and atherosclerosis (20). To understand the etiology of these disorders, it is essential to
elucidate the molecular mechanisms whereby hyperplasia is induced in
VSMC. It is now believed that VSMC proliferation is suppressed under
physiological conditions but can be rapidly restimulated by growth
factors, such as platelet-derived growth factor (PDGF) and fibroblast
growth factor, in some pathological and adaptive states, resulting in
vascular hyperplasia (29). On the other hand, if VSMC fail to divide,
cell size is considerably increased without increment in cell number
(hypertrophy). The role of vasoconstrictive hormones, such as
angiotensin II (ANG II) and arginine vasopressin (AVP), in these
processes still remains controversial. For instance, ANG II was
reported to show mitogenic effects on some but not all VSMC lines from
Wistar-Kyoto rats (37). Daemen et al. (6) described that ANG II
treatment resulted in a marked enhancement of VSMC proliferation at the
injury sites of blood vessels. In contrast, other studies indicated
that ANG II induced only hypertrophy in cultured VSMC (2). Geisterfer
et al. (14) reported that ANG II had no detectable mitogenic activity
in VSMC and arrested cells at the
G2 phase of the cell cycle, which
may account for vascular hypertrophy and hyperploidy. The
growth-regulating effect of AVP on VSMC has not been well studied,
although it has been shown that AVP stimulates mitogen-activated
protein (MAP) kinase, an important mediator of cell proliferation (17,
34). Further investigation is required to clarify the role of
vasoconstrictive hormones in growth regulation of VSMC.
The division of eukaryotic cells is regulated at
G1/S and
G2/M boundaries by a family of
protein kinases collectively known as cyclin-dependent kinases (cdks)
(10, 26). D-type cyclins are induced in quiescent cells in response to
growth factors or hormones and activate cdk4 and cdk6 to phosphorylate
retinoblastoma (RB) family proteins (pRB, p107, and p130), thereby
allowing cells to progress through the
G1 phase (21). Subsequently,
active cdk2/cyclin E complex is formed to regulate the final step of G1/S transition (30). During the S
phase of the cell cycle, cdk2/cyclin A complex is continuously present
and participates in the initiation and maintenance of DNA synthesis
(15). It has been demonstrated that cdc2 kinase is crucial for entry
into mitosis (9, 13). In the G2
phase, cdc2/cyclin B complex receives the signal that DNA replication
has been satisfactorily completed and phosphorylates various
cytoskeletal and nuclear proteins, such as histone H1 and lamins, whose
phosphorylation is essential for
G2/M transition and mitosis (25).
Recent studies have shown that cdks are also involved in cell cycle
regulation of VSMC as demonstrated in other cell types (1, 27, 48).
The present study was therefore undertaken to study the effects of ANG
II and AVP on the cell cycle profile of cultured rat VSMC to reveal
whether these hormones have proliferative effects on VSMC under
physiological conditions. Furthermore, we examined the effects of ANG
II and AVP on cell cycle regulatory elements, such as cdk2 and cdc2, to
elucidate the molecular basis of growth regulation of VSMC by
vasoconstrictive hormones.
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MATERIALS AND METHODS |
Preparation of cells and cell culture.
Rat VSMC were isolated as described in our previous studies
(31-33). Briefly, thoracic aortas were dissected from 8-10
male Sprague-Dawley rats (200-300 g) and then incubated in MEM
containing 2 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ)
for 1 h at 37°C. After incubation, aortas were minced and incubated again in MEM containing 2 mg/ml collagenase for 2 h at 37°C. The dispersed cells were resuspended in MEM, pH 7.4, containing 1 µM
L-glutamine, 100 U/ml
penicillin, and 10% FCS. The cells were kept in a humidified incubator
under 95% air-5% CO2 at 37°C
and subcultured with 0.25% trypsin-0.1 mM EGTA treatment. After a day
of subculture, the cells were synchronized for 24 h in serum-free MEM
to obtain quiescent cells (34). The medium was then replaced with MEM
containing effectors, and culture was continued for given culture periods.
Measurement of [3H]thymidine
incorporation.
A [3H]thymidine
incorporation assay was performed as described previously (18) with
minor modifications. Cells were grown in 24-well culture clusters for
indicated periods and further incubated with 1 µCi/well of
[3H]thymidine
(specific activity 80.8 Ci/mmol; NEN, Wilmington, MA) for an additional
4 h. Thereafter, the cells were washed twice with PBS and lysed with
0.2 N NaOH, and the lysates were collected into counting vials
containing 5 ml of scintillation solution. Radioactivity was measured
using a liquid scintillation counter (Aloka LSC-671, Tokyo, Japan).
Cell cycle analysis.
Cells were resuspended in 0.5 ml of propidium iodide solution [50
µg/ml propidium iodide (Sigma, St. Louis, MO) in 0.1% sodium citrate
and 0.1% Nonidet P-40] and incubated for 15 min at 4°C. DNA
content was then analyzed by flow cytometry with the FACScan/CellFIT system (Becton-Dickinson, San Jose, CA).
Western blotting.
Cells were washed with ice-cold Tris-buffered saline (TBS) [25 mM
Tris · HCl (pH 8.0) and 150 mM NaCl] and lysed
for 30 min at 4°C with lysis buffer [50 mM
Tris · HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40,
100 mM sodium fluoride, and 200 µM sodium orthovanadate] containing 10 µg each of aprotinin, phenylmethylsulfonyl fluoride, and leupeptin (Sigma). Cell lysates were centrifuged for
15 min, and the supernatants (40 µg/sample) were applied onto 10%
SDS-polyacrylamide gels. After electrophoresis, proteins were
transferred onto nitrocellulose membranes (Amersham, Amersham, UK) in
transfer buffer containing 25 mM Tris · HCl, 192 mM
glycine, and 20% methanol. Residual binding sites on the membrane were
blocked with 5% skim milk in TBS for >30 min at room temperature.
The blots were incubated with 1 µg/ml primary antibodies overnight at
4°C, washed with TBS containing 0.05% Tween 20 three times (5 min
each), and probed with a 1:1,000 dilution of goat anti-mouse
horseradish peroxidase-conjugated antibody for 40 min at room
temperature. After the blots were washed, they were incubated with the
enhanced chemiluminescence substrate and exposed to Kodak X-Omat film
(Eastman Kodak, Rochester, NY) for visualization of immunoreactive
bands. For reprobing, antibodies on the blots were stripped by
incubation in 62.5 mM Tris · HCl (pH 6.8), 2% SDS,
and 100 mM 2-mercaptoethanol at 50°C for 30 min.
Specific monoclonal antibodies used in this study were as follows:
anti-cdk2 (clone 55; Transduction Laboratories, Lexington, KY),
anti-proliferating cell nuclear antigen (PCNA; clone 5A10; MBL, Nagoya,
Japan), anti-cdc2 (clone 1; Transduction Laboratories), anti-cyclin B1
(GNS1; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-cyclin E
(M-20; Santa Cruz Biotechnology).
Histone H1 kinase assay.
Histone H1 kinase activity was measured according to the standard
method (22). Whole cell lysates were prepared as described in
Western blotting, and 250 µg of each sample were subjected to immunoprecipitation with either
anti-cdk2 or anti-cdc2 antibody. Immune complexes were collected on
protein G-Sepharose (Pharmacia), washed vigorously with lysis buffer,
and resuspended in 20 µl of reaction mixture containing 20 mM
Tris · HCl, pH 7.4, 10 mM MgCl2, 4.5 µM
-mercaptoethanol, 1 mM EGTA, 50 µM ATP, 0.2 µCi [
-32P]ATP, and 0.1 mg/ml histone H1 (type III-S, Sigma). Incubation was carried out for 30 min at 30°C and terminated by the addition of sample loading buffer
and boiling. The samples were analyzed by 12% SDS-PAGE and autoradiography.
Northern blotting.
Total cellular RNA was isolated by cesium chloride ultracentrifugation.
RNA samples (10 µg each) were electrophoresed in a 1.0% agarose gel
containing 6% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, and 1 mM
EDTA and then blotted onto synthetic nylon membranes. The membranes
were hybridized with cdc2 cDNA probe, which was labeled with
[32P]dCTP (NEN) with
the oligonucleotide random priming method. A 405-bp fragment, spanning
nucleotides 46 to 450 of mouse cdc2 cDNA (42), was generated by RT-PCR
and used as a probe.
Transient transfection and chloramphenicol acetyltransferase assay.
Plasmids were introduced into VSMC by the calcium phosphate
precipitation method (16). Cells were left with DNA precipitates for 16 h in the presence of 10% FCS, washed with PBS, and cultured in the
presence of 10% FCS or with vasoconstrictive hormones in FCS-free
medium for 48 h.
As previously reported (12), a 5'-untranslated sequence of the
cdc2 promoter up to nucleotide 
383 relative to the transcription start site was linked to the bacterial chloramphenicol
acetyltransferase (CAT) gene in pCAT-basic vector (Promega, Madison,
WI), and this vector was used as a reporter plasmid (designated
pCAT-cdc2). pCAT-control vector (Promega), which contains SV40 promoter
and enhancer sequences, was transfected into cells in another dish, and
the cells were cultured under the same conditions. pSV--gal vector
(Promega) was cotransfected (2 µg/dish) with test plasmids (18 µg/dish) to monitor transfection efficiencies of each sample. All
plasmids were purified twice by cesium chloride gradient
ultracentrifugation and ethanol precipitation before transfection.
CAT activities were measured quantitatively by liquid scintillation
counting and were corrected by -galactosidase activities and protein
contents (40). Each result was adjusted according to the value obtained
with the transfection of pCAT-control vector into corresponding cells
and expressed as the ratio of pCAT-cdc2 to pCAT-control (relative CAT activity).
Gel retardation assay.
Nuclear extract was prepared according to the method of Dignam et al.
(8). Samples (5 µg) were incubated with ~0.5 ng (10,000 counts/min)
of 32P-labeled oligonucleotide
probe in the presence of 1 µg of sonicated salmon sperm DNA in a
final volume of 25 µl. Incubations were carried out at room
temperature for 30 min in 20 mM HEPES, pH 7.9, 0.1% Nonidet P-40, 40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA,
0.5 mM dithiothreitol, and 10% glycerol. The DNA-protein complexes were resolved on a 4% polyacrylamide gel (0.15 × 16 × 20 cm; 86:1 wt/wt ratio of acrylamide to bisacrylamide) in 0.25×
Tris-borate-EDTA buffer at 4°C.
Double-stranded oligonucleotides containing the putative E2F
binding site
(5'-TCCGCTC
TCTGCACTC-3', nucleotides
139 to 114) and the CDE/CHR region
(5'GA-GCTTT
ACTGCTGG-3', nucleotides 32 to 1) of the rat cdc2 promoter
(41) were used as probes in this study (consensus sequences are
underlined). Cold competition assays and antibody perturbation
experiments were carried out as previously described (19) to identify
the nature of each complex. The following antibodies were used (all from Santa Cruz Biotechnology except as noted): anti-E2F-1, C-20; anti-E2F-2, C-20; anti-E2F-3, C-18; anti-E2F-4, C-108; anti-E2F-5, E-19; anti-pRB, C36 (Pharmingen); anti-p130, clone 10 (Transduction Laboratories); anti-p107, SD9; and anti--actin, Ab-1 (Oncogene Science, Uniondale, NY).
Statistical analysis.
The results are expressed as means ± SE of the samples, which
represented at least three separate experiments. The unpaired Student's t-test and one-way ANOVA
combined with Scheffé's test were used for statistical
comparisons. A P value <0.05 was
considered to be statistically significant.
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RESULTS |
Effects of FCS and the vasoconstrictive hormones AVP and ANG II on
DNA synthesis and cell cycle distribution of cultured rat VSMC.
In an attempt to examine the effects of FCS and vasoconstrictive
hormones on VSMC, we first determined the optimal concentration of each
effector to induce maximal stimulation of DNA synthesis. Rat VSMC were
starved for 24 h in serum-free medium to induce quiescence and were
restimulated by the addition of various doses of FCS, AVP, and ANG II.
[3H]thymidine
incorporation, well correlated with cellular DNA synthesis in S phase,
was assayed after 12 h of culture as described in MATERIALS AND METHODS. As shown in
Fig. 1A,
maximal uptake of [3H]thymidine was
observed at the concentrations of 10%, 1 µM, and 0.1 µM for FCS,
AVP, and ANG II, respectively. On the basis of these results, we
decided to use 10% FCS, 1 µM AVP, and 0.1 µM ANG II as optimal
concentrations in subsequent experiments. We next examined the kinetics
of [3H]thymidine
incorporation of rat VSMC cultured with FCS, AVP, and ANG II. The
optimal concentrations of each effector induced a time-dependent
increase in
[3H]thymidine
incorporation with a peak at 12 h, although the maximal level of
[3H]thymidine uptake
was significantly higher in FCS-treated than in hormone-treated cells
(Fig. 1B).
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Fig. 1.
Effects of FCS, arginine vasopressin (AVP), and angiotensin II (ANG II)
on [3H]thymidine
incorporation of vascular smooth muscle cells (VSMC) in culture. Rat
VSMC were incubated for 24 h in serum-free medium to obtain quiescent
cells. Cells were then exposed to each effector at various
concentrations for 12 h (A) or
incubated with each effector at its optimal concentration for indicated
periods (B). Values were adjusted
according to protein contents in each sample. Bars represent means ± SE (n = 5); cpm, counts/min.
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The changes in cell cycle distribution were monitored by serial
analysis of the DNA histogram of VSMC cultured with FCS, AVP, and ANG
II (Table 1). Approximately 75% of
serum-starved cells were arrested in the
G0/G1
phase of the cell cycle (Table 1) with only 5.1% in S phase. No
significant change in the cell cycle profile was observed in VSMC
cultured in serum-free medium for 12 and 24 h (Table 1). However,
readdition of 10% FCS resulted in a marked increase in S phase (from
5.1% at 0 h to 39.9% at 12 h). The treatment of VSMC with AVP and ANG
II also increased the proportion of cells in S phase, but the level of
increase was remarkably lower than that with FCS treatment (12.9% for
AVP and 12.4% for ANG II). After S phase entry, FCS-treated cells were
able to progress through the G2
phase to the M phase (27.6% at 24 h), resulting in an increase in cell
number. In contrast, neither AVP nor ANG II induced
G2/M phase transition (14.7 and 16.4% at 24 h, respectively) or increased the cell number. To convincingly demonstrate that AVP and ANG II did not induce mitosis of
VSMC, we examined the morphological changes during culture under
phase-contrast microscopy. As is clearly shown in Fig.
2, cell proliferation was not observed in
VSMC cultured with AVP and ANG II, whereas FCS provoked a striking
increase in cell number after 24 h. It is of note that cell size was
considerably increased in hormone-treated cells. These findings
indicate that the vasoconstrictive hormones AVP and ANG II are capable
of promoting S phase entry in quiescent VSMC but fail to induce further
progression into M phase and cell division. On the other hand, FCS has
much stronger effects on cell cycle progression, which allow cells to
transverse the G2/M boundary and
increase in number.
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Fig. 2.
Effects of FCS, AVP, and ANG II on cell growth of rat VSMC. After 24-h
serum deprivation, rat VSMC were cultured with 10% FCS, 1 µM AVP,
and 0.1 µM ANG II for 3 days. Phase-contrast micrographs were taken
after indicated incubation times.
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Effects of FCS and the vasoconstrictive hormones AVP and ANG II on
cell cycle regulatory elements.
To clarify the molecular basis of the differential effects of FCS and
vasoconstrictive hormones on the growth of VSMC, we screened the
expression of some cell cycle regulatory elements, including major
cyclin-dependent kinases (cdk2 and cdc2), PCNA, and major cyclins
(cyclins E and B1) by Western blotting. As shown in Fig.
3, expression of cdk2, cyclin E, and PCNA,
all of which are implicated in
G1/S transition and DNA
replication, increased in a time-dependent manner in FCS-treated VSMC.
The increase began after 2 h of treatment with FCS and continued up to
24 h (for quantitation, see Table 2). This
is consistent with the results of
[3H]thymidine
incorporation and cell cycle analysis shown in Fig. 1B and Table 1. Similar results were
obtained in the cells treated with AVP and ANG II in agreement with
their ability to induce S phase entry, although the level of increase
was weaker than that of FCS for cdk2 and PCNA (Table 2). We next
investigated the expression of cdc2 and cyclin B1, both of which are
critical regulators for entry into mitosis, using the same samples
(Fig. 3, A and
B). FCS also induced a
time-dependent increase in the amounts of cdc2 and cyclin B1; the
increase occurred 12 h after addition of FCS, which was later than that
of cdk2, cyclin E, and PCNA, compatible with their roles in the cell
cycle. In contrast, vasoconstrictive hormones failed to upregulate cdc2
protein; rather, the amounts of cdc2 gradually decreased in VSMC
cultured with AVP and ANG II (Table 2). Similarly, cyclin B1 induction
was not observed in hormone-treated cells.
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Fig. 3.
Effects of FCS, AVP, and ANG II on cell cycle regulatory proteins.
Serum-starved rat VSMC were cultured with each effector at its optimal
concentration for indicated periods and subjected to Western blot
analysis for expression of cdk2, proliferating-cell nuclear antigen
(PCNA), and cdc2 (A) and cyclins E
and B1 (B). Cdk2 and cdc2 were
migrated as doublets of 34 and 33 kDa, and cyclin E was discernible as
2 distinct bands of 50 and 42 kDa.
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We next investigated the changes of cdk2 and cdc2 kinase activities in
these cells. Serum-starved VSMC were cultured in the presence of FCS,
AVP, and ANG II, and whole cell lysates were isolated after 12 and 24 h
and subjected to immunoprecipitation with anti-cdk2 and -cdc2
antibodies, followed by histone H1 kinase assay. Consistent with the
results of Western blot analysis, both FCS and the vasoconstrictive
hormones induced an increase in cdk2 kinase activity (data not shown).
However, cdc2 kinase activity was upregulated in FCS-treated cells but
not in hormone-treated cells (Fig. 4). The
absence of cdc2 induction and activation may account for the failure of
these hormones to promote progression through the
G2 phase to the M phase.
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Fig. 4.
Histone H1 kinase activity of VSMC treated with FCS and ANG II.
Serum-starved rat VSMC were cultured with either 10% FCS or 0.1 µM
ANG II for  24 h. Whole cell lysates were prepared at indicated time
points and subjected to immunoprecipitation with anti-cdc2-specific
antibody, followed by histone H1 kinase assay.
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Effects of FCS and the vasoconstrictive hormones AVP and ANG II on
cdc2 mRNA expression and the cdc2 promoter.
To understand the mechanisms of the absence of cdc2 protein induction
by AVP and ANG II, we investigated cdc2 mRNA expression in cultured rat
VSMC by Northern blotting. As shown in Fig.
5, cdc2 mRNA was not detectable in
serum-starved quiescent VSMC, consistent with previous reports (28).
FCS readily induced authentic 1.8-kb cdc2 transcript after 12 h of
treatment (Fig. 5, lanes 2 and
3), whereas cdc2 mRNA expression was
absent in cells treated with AVP and ANG II at their optimal
concentrations. This result indicates that the lack of cdc2 induction
by these hormones is primarily caused at the level of transcription.
Therefore, we examined the effects of FCS and vasoconstrictive hormones
on cdc2 promoter activity by transient CAT assay. We used the pCAT-cdc2 construct, which contains a 5'-untranslated region of the cdc2 gene (up to 383), as a reporter plasmid. This segment was
previously shown to possess a full promoter activity for
transactivation of the cdc2 gene (11, 12, 41). pCAT-control vector,
which possesses strong SV40 promoter with enhancer, was transfected into cells in another dish in parallel to obtain relative CAT activity,
which was expressed as the ratio of pCAT-cdc2 to pCAT-control (Fig.
6). As expected, CAT activity was
significantly high in rat VSMC cultured with 10% FCS
(P < 0.01), whereas AVP and ANG II
failed to activate the cdc2 promoter. This result explains the
difference of cdc2 mRNA expression between FCS-treated cells and
vasoconstrictive hormone-treated cells.
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Fig. 5.
Expression of cdc2 mRNA in VSMC treated with FCS, AVP, and ANG II.
Total cellular RNA was isolated from cells cultured with each effector
at indicated time points. Northern blot analysis was performed with
mouse cdc2 cDNA as a probe. Positions of rRNA (28S, 18S) and cdc2
transcript (1.8 kb) are shown at left.
Ethidium bromide-staining of 28S and 18S rRNA is shown as a loading
control (bottom).
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Fig. 6.
Effects of FCS, AVP, and ANG II on cdc2 promoter activity. Rat VSMC
were transfected with pCAT-cdc2 reporter plasmid, containing a
5'-untranslated sequence of cdc2 gene (up to 383
position), and pCAT-control vector (positive control) and cultured with
each effector for 48 h. pSV--gal vector was cotransfected with test
plasmids to monitor transfection efficiencies of each dish.
Chloramphenicol acetyltransferase (CAT) activity in each extract was
corrected by -galactosidase activity and protein content. Relative
CAT activity was shown as ratio of values obtained with pCAT-cdc2 and
pCAT-control vectors (pCAT-cdc2/pCAT-control). Bars represent means ± SE (n = 4).
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Finally, we examined the molecular basis of why AVP and ANG II fail to
activate the cdc2 promoter. The promoter construct used in this study
contains the putative E2F-binding site at the nucleotide positions
230 to 223 and the CDE/CHR region at the nucleotide
positions 24 to 6. Recent studies have indicated that the
former is involved in both positive and negative regulation and the
latter is responsible for repression of the cdc2 promoter. Binding of
E2F transcription factors complexed with RB family proteins, such as
E2F-4/p130 and E2F-1/pRB, at the E2F-binding site suppresses
transcription of the cdc2 gene (7, 50), and this repression is released
as RB family proteins are phosphorylated by cdk4/cyclin D and
cdk2/cyclin E complexes (21). After the release of E2F/RB family
protein complexes, free E2F (mainly E2F-1) occupies this site and
facilitates cdc2 transcription (12, 41). Bearing this background in
mind, we carried out gel retardation assays to determine the factor(s)
bound to the E2F-binding site and the CDE/CHR region in VSMC cultured
with FCS, AVP, and ANG II. Nuclear extracts were isolated from the
cells after 0, 12, and 24 h of culture and incubated with
32P-labeled oligonucleotide
corresponding to the E2F-binding site of rat cdc2 promoter. DNA-protein
complexes were resolved on native polyacrylamide gels. A representative
result is shown in Fig. 7. Specificity of
these complexes was verified by cold competition assay (data not
shown). The identity of each complex was determined by antibody
perturbation experiments using specific antibodies against E2F and RB
family proteins (Fig. 7B): signal
intensity of the fastest migrating band was diminished by anti-E2F-1
but not by any other antibodies, indicating that it represents free E2F-1; the slowest migrating band was completely supershifted by
anti-E2F-4 and diminished by anti-pRB, showing that it represents E2F-4/pRB complex; and the bands with intermediate mobilities were
affected by anti-pRB and anti-p130, suggesting that these represent E2F
complexes containing pRB and p130. In quiescent VSMC, E2F/RB family
protein complexes (E2F-4/pRB, E2F/p130, and E2F/pRB) were dominant and
transcriptionally active free E2F-1 was not present, consistent with
the lack of cdc2 mRNA expression (Fig.
7A, lanes
1, 4, and
7). After 12 h of culture with FCS, the amounts of E2F/p130 and E2F/pRB decreased and free E2F-1 binding was readily induced, which is accompanied by cdc2 mRNA expression (Fig.
7A, lanes
2 and 3). When VSMC
were treated with AVP, the amounts of E2F/p130 and E2F/pRB complexes
were reduced as in FCS-treated cells, but free E2F-1 did not appear
(Fig. 7A, lanes
4 and 5). ANG II
treatment showed similar effects (Fig.
7A, lanes
8 and 9). These
results suggest that the absence of free E2F-1 binding to the cdc2
promoter is at least in part responsible for the lack of cdc2
transcription in hormone-treated cells. On the other hand, DNA-protein
complex was not detectable with oligonucleotide probe corresponding to
the CDE/CHR region of rat cdc2 promoter (data not shown). Thus the role
of the CDE/CHR region in the regulation of rat cdc2 promoter remains to
be clarified in VSMC.
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Fig. 7.
Effects of FCS, AVP, and ANG II on E2F complexes on cdc2 promoter.
A: nuclear extracts were isolated from
rat VSMC cultured with each effector at indicated time points and
incubated with 32P-labeled
oligonucleotides containing putative E2F-binding site of rat cdc2
promoter. DNA-protein complexes were resolved on 4% polyacrylamide
gels. Positions of specific E2F complexes are indicated at
left. Bands indicated by stars were
found to be nonspecific by cold competition assay.
B: identity of each complex
(E2F-4/pRB, E2F/p130, E2F/pRB, and free E2F-1) was determined by
perturbation experiments using HeLa cell nuclear extracts and
antibodies against E2F and retinoblastoma (RB) family proteins as
previously described (28, 31). Arrows indicate supershifted bands.
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DISCUSSION |
Previous studies have suggested that ANG II and AVP are
vasoconstrictive hormones as well as possible growth factors for VSMC (6, 36, 37). However, the effects of ANG II and AVP on the
proliferation of VSMC still remain controversial. Moreover, there is
little information about the role of these hormones in cell cycle
regulation of VSMC. In the present work, we attempted to address two
questions regarding the growth-related effects of ANG II and AVP on
VSMC. First, do these hormones promote the proliferation of VSMC under
physiological conditions, and second, do these hormones modulate the
cell cycle regulatory elements of VSMC? In contrast to ANG II and AVP,
FCS has been shown to contain potent growth factors, such as PDGF and
fibroblast growth factor, and can induce proliferation in cultured VSMC
(29, 48). Therefore, in this study, investigations were carried out to
compare the growth-promoting effects of FCS with those of ANG II and AVP.
First, we examined the effects of ANG II and AVP on DNA synthesis and
cell cycle distribution of rat VSMC by
[3H]thymidine
incorporation and flow cytometry.
[3H]thymidine
incorporation assay revealed that all effectors were able to stimulate
DNA synthesis in quiescent VSMC in a time-dependent manner. The
increase of S phase in cells treated with each effector was confirmed
by analyzing DNA histograms by flow cytometry, although the S
phase-inducing effect was remarkably weaker with AVP and ANG II than
with FCS. Furthermore, it is of note that both AVP and ANG II failed to
promote progression through the G2
phase to the M phase, in contrast to FCS, which showed a strong
mitogenic effect on VSMC. No increase in cell number of hormone-treated VSMC was confirmed by morphological examination under phase-contrast microscopy. These findings suggest that the vasoconstrictive hormones AVP and ANG II do not induce hyperplasia of VSMC under physiological conditions. This is in good agreement with some but not all previous studies on this subject (2, 14). However, it was reported that ANG II
induced cell proliferation at the injured sites of blood vessels,
suggesting that these hormones can act as mitogens under certain
conditions (6, 27). Because ANG II is known to stimulate the secretion
of autocrine growth factors, such as PDGF and transforming growth
factor-1, from VSMC (28, 29), the mitogenic effect of
vasoconstrictive hormones may be indirect and is mediated by these
factors. Further investigation is required to clarify the roles of AVP
and ANG II in vascular hyperplasia associated with pathological states
such as hypertension or atherosclerosis.
We next attempted to clarify the mechanisms whereby vasoconstrictive
hormones induce G1/S transition
but fail to promote further progression through the
G2 phase to the M phase. Because
both hypertrophic and hyperplastic (mitogenic) stimuli share the early cell signaling in VSMC, such as activation of MAP kinase, AP-1, c-myc, and
p21ras (17, 28, 34, 39), it is
important to determine the downstream events responsible for different
final responses. In a rat carotid artery balloon injury model, the
proliferation of VSMC was shown to be regulated by cell cycle
regulatory elements including cdk2, cdc2, PCNA,
p21cip1/waf1, and
p27kip1 (1, 4, 5, 27). To
elucidate the molecular basis of different growth responses of VSMC to
FCS and vasoconstrictor hormones, we examined their effects on the cell
cycle control machinery: primary regulators of
G1/S transition (cdk2 and cyclin E), DNA replication (PCNA), and
G2/M transition (cdc2 and cyclin B).
Western blot analysis revealed that FCS as well as AVP and ANG II
increased the expression of cdk2, cyclin E, and PCNA, consistent with
their ability to induce G1/S
transition and DNA synthesis in quiescent VSMC. In contrast, only FCS
was capable of inducing cdc2/cyclin B complex in VSMC. These findings
indicate that cdc2 and cyclin B are the key elements in determining the
response of VSMC to different stimuli and that the failure of
G2/M transition in hormone-treated
cells is attributable to the lack of cdc2 and cyclin B induction. The
absence of cdc2 induction by vasoconstrictive hormones was found to be
at the level of transcription. We therefore sought to determine how FCS
and vasoconstrictive hormones differentially regulate the cdc2
promoter. Regulation of the cdc2 promoter has been a subject of
extensive investigations, and some critical components have been
defined. Because previous studies indicated that a
5'-untranslated sequence of the cdc2 gene up to 383 was sufficient to activate transcription of both human and rat cdc2 promoters (11, 12, 41), we used a reporter plasmid containing this
segment to examine the effects of FCS and vasoconstrictive hormones.
Transient transfection-CAT assays revealed that AVP and ANG II failed
to activate the cdc2 promoter, whereas FCS induced significant
transactivation of cdc2 in VSMC. This clearly explains why cdc2 mRNA
expression was not inducible by these hormones. We then explored the
mechanisms of insufficient activation of the cdc2 promoter by AVP and
ANG II. It has been shown that cdc2 promoter is positively regulated by
E2F (especially free E2F-1) (12, 41), nuclear factor-Y (24),
c-myb (23), ets2 (49), c-myc (3), and activated N-ras (3) and
negatively regulated by E2F/RB family protein complexes (7, 45, 50),
the CDE/CHR region (the R box) (43, 51), and the upstream
negative-regulatory elements (11, 46). The promoter segment used in our
study contains the putative E2F-binding site, which is involved in both positive and negative regulation (at 230 to 223), and the
CDE/CHR region, which is responsible for repression of the cdc2
promoter (at 24 to 6). We examined binding of
transcription factors to these sites by gel retardation assays with
oligonucleotides containing each sequence as probes. Binding of the
E2F/RB family protein complexes E2F/p130 and E2F/pRB was observed at
the E2F-binding site when VSMC were in a quiescent state and did not
express cdc2 mRNA. This is compatible with the properties of E2F/RB
family protein complexes as transcriptional repressors for cdc2 (7, 45,
50). These complexes disappeared on stimulation of the cells with FCS,
AVP, and ANG II, probably because of phosphorylation of pRB and p130 by
cyclin D-dependent kinases activated by these effectors. In support of
this notion, Watanabe et al. (47) reported evidence that ANG II
activated cyclin D-dependent kinases through p21ras/ERK/AP-1 pathways. However,
the disappearance of repressor complexes seemed not to be sufficient
for cdc2 promoter activation. Induction of cdc2 promoter was
accomplished with FCS, but not with AVP and ANG II, through binding of
free (uncomplexed) E2F-1 to the E2F sites. On the other hand, the
significance of the CDE/CHR region was not demonstrated in our study,
because no protein binding was detected at this site. These results
suggest that recruitment of free E2F-1 is necessary for transcriptional
activation of the cdc2 gene in VSMC. Failure of induction of cdc2 by
ANG II was also observed in neonatal cardiac myocytes, which may
contribute to irreversible cell cycle arrest of cardiocytes (38).
Conversely, the importance of free E2F-1 binding for cdc2 promoter
activation was demonstrated in the growth enhancement of osteoblast
precursors by parathyroid hormone, as observed in FCS-treated VSMC
(35). Furthermore, it is possible that the lack of cyclin B1 induction by vasoconstrictive hormones is mediated through the similar mechanisms involving E2F, because cyclin B1 promoter is also under the control of
E2F (44).
In summary, we demonstrate that AVP and ANG II can promote
G1/S transition and DNA synthesis
but do not induce G2/M transition and mitosis, primarily because of the failure of cdc2 mRNA induction. The failure of cdc2 promoter activation is at least in part
attributable to defective binding of free E2F-1 to the promoter. We are
currently trying to identify the mechanisms of defective binding of
E2F-1 to the cdc2 promoter in hormone-treated cells. This system may serve as the major mechanism underlying hormone-induced hypertrophy of VSMC.
| |
ACKNOWLEDGEMENTS |
This work was supported in part by Grants-in-Aid from the Ministry
of Education, Science and Culture of Japan.
| |
FOOTNOTES |
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.
Address for reprint requests and other correspondence: K. Okada, Div.
of Endocrinology and Metabolism, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan
(E-mail: furuyu{at}ms.jichi.ac.jp).
Received 1 October 1998; accepted in final form 23 March 1999.
| |
REFERENCES |
1.
Abe, J.,
W. Zhou,
J. Taguchi,
N. Takuwa,
K. Miki,
H. Okazaki,
K. Kurokawa,
M. Kumada,
and
Y. Takuwa.
Suppression of neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2 oligonucleotides in rat carotid artery.
Biochem. Biophys. Res. Commun.
198:
16-24,
1994[Medline].
2.
Berk, B. C.,
V. Vekshtein,
H. M. Gordon,
and
T. Tsuda.
Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells.
Hypertension
13:
305-314,
1989[Abstract/Free Full Text].
3.
Born, T. L.,
J. A. Frost,
A. Schonthal,
G. C. Prendergast,
and
J. R. Feramisco.
c-Myc cooperates with activated Ras to induce the cdc2 promoter.
Mol. Cell. Biol.
14:
5710-5718,
1994[Abstract/Free Full Text].
4.
Chang, M. W.,
E. Barr,
M.-M. Lu,
K. Barton,
and
J. M. Leiden.
Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty.
J. Clin. Invest.
96:
2260-2268,
1995.
5.
Chen, D.,
K. Krasinski,
D. Chen,
A. Sylvester,
J. Chen,
P. D. Nisen,
and
V. Andres.
Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27KIP1, an inhibitor of neointima formation in the rat carotid artery.
J. Clin. Invest.
99:
2334-2341,
1997[Medline].
6.
Daemen, M. J. A. P.,
D. M. Lombardi,
F. T. Bosman,
and
S. M. Schwartz.
Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall.
Circ. Res.
68:
450-456,
1991[Abstract/Free Full Text].
7.
Dalton, S.
Cell cycle regulation of the human cdc2 gene.
EMBO J.
11:
1797-1804,
1992[Medline].
8.
Dignam, J. D.,
R. M. Lebovitz,
and
R. G. Roeder.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:
1475-1489,
1983[Abstract/Free Full Text].
9.
Dunphy, W. G.,
L. Brizuela,
D. Beach,
and
J. Newport.
The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis.
Cell
54:
423-431,
1988[Medline].
10.
Furukawa, Y.
Cell cycle regulation of hematopoietic stem cells.
Hum. Cell
11:
81-92,
1998[Medline].
11.
Furukawa, Y.,
S. Iwase,
Y. Terui,
J. Kikuchi,
T. Sakai,
M. Nakamura,
S. Kitagawa,
and
M. Kitagawa.
Transcriptional activation of the cdc2 gene is associated with Fas-induced apoptosis of human hematopoietic cells.
J. Biol. Chem.
271:
28469-28477,
1996[Abstract/Free Full Text].
12.
Furukawa, Y.,
Y. Terui,
K. Sakoe,
M. Ohta,
and
M. Saito.
The role of cellular transcription factor E2F in the regulation of cdc2 mRNA expression and cell cycle control of human hematopoietic cells.
J. Biol. Chem.
269:
26249-26258,
1994[Abstract/Free Full Text].
13.
Gautier, J.,
C. Norbury,
M. Lohka,
P. Nurse,
and
J. Maller.
Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+.
Cell
54:
433-439,
1988[Medline].
14.
Geisterfer, A. A. T.,
M. J. Peach,
and
G. K. Owens.
Angiotensin II induces hypertrophy not hyperplasia of cultured rat aortic smooth muscle cells.
Circ. Res.
62:
749-756,
1988[Abstract/Free Full Text].
15.
Girard, F.,
U. Strausfeld,
A. Fernandez,
and
N. J. C. Lamb.
Cyclin A is required for the onset of DNA replication in mammalian fibroblasts.
Cell
67:
1169-1179,
1991[Medline].
16.
Gorman, C. M.,
L. F. Moffat,
and
B. H. Howard.
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol. Cell. Biol.
2:
1044-1051,
1982[Abstract/Free Full Text].
17.
Granot, Y.,
E. Erikson,
H. Fridman,
V. Van Putten,
B. Williams,
R. W. Schrier,
and
J. L. Maller.
Direct evidence for tyrosine and threonine phosphorylation and activation of mitogen-activated protein kinase by vasopressin in cultured rat vascular smooth muscle cells.
J. Biol. Chem.
268:
9564-9569,
1993[Abstract/Free Full Text].
18.
Guo, X.,
K. Okada,
N. Fujita,
S. Ishikawa,
N. Komatsu,
and
T. Saito.
Inhibitory effect of BQ-123 on endothelin-1-stimulated mitogen-activated protein kinase and cell growth of rat vascular smooth muscle cells.
Hypertens. Res.
19:
23-30,
1996[Medline].
19.
Iwase, S.,
Y. Furukawa,
J. Kikuchi,
M. Nagai,
Y. Terui,
M. Nakamura,
and
H. Yamada.
Modulation of E2F activity is linked to interferon-induced growth suppression of hematopoietic cells.
J. Biol. Chem.
272:
12406-12414,
1997[Abstract/Free Full Text].
20.
Jackson, C. L.,
and
S. M. Schwartz.
Pharmacology of smooth muscle cell replication.
Hypertension
20:
713-736,
1992[Abstract/Free Full Text].
21.
Kato, J.,
H. Matsushime,
S. W. Hiebert,
M. E. Ewen,
and
C. J. Sherr.
Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.
Genes Dev.
7:
331-342,
1993[Free Full Text].
22.
Kitagawa, M.,
T. Okabe,
H. Ogino,
H. Matsumoto,
I. Suzuki-Takahashi,
T. Kokubo,
H. Higashi,
S. Saitoh,
Y. Taya,
H. Yasuda,
Y. Ohba,
S. Nishimura,
N. Tanaka,
and
A. Okuyama.
Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase.
Oncogene
8:
2425-2432,
1993[Medline].
23.
Ku, D.-H.,
S.-C. Wen,
A. Engelhard,
N. C. Nicolaides,
K. E. Lipson,
T. A. Marino,
and
B. Calabretta.
c-myb transactivates cdc2 expression via Myb binding sites in the 5'-flanking region of the human cdc2 gene.
J. Biol. Chem.
268:
2255-2259,
1993[Abstract/Free Full Text].
24.
Liu, Q.,
H. Yan,
N. J. Dawes,
Y. Lu,
and
H. Zhu.
Transcriptional activation of the p34cdc2 gene by cdc2 promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.
Circ. Res.
82:
251-260,
1998[Abstract/Free Full Text].
25.
Moreno, S.,
and
P. Nurse.
Substrates for p34cdc2: in vivo veritas?
Cell
61:
549-551,
1990[Medline].
26.
Morgan, D. O.
Cyclin-dependent kinases: engines, clocks, and microprocessors.
Ann. Rev. Cell Dev. Biol.
13:
261-291,
1997[Medline].
27.
Morishita, R.,
G. H. Gibbons,
K. E. Ellison,
M. Nakajima,
L. Zhang,
Y. Kaneda,
T. Ogihara,
and
V. J. Dzau.
Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia.
Proc. Natl. Acad. Sci. USA
90:
8474-8478,
1993[Abstract/Free Full Text].
28.
Naftilan, A. J.,
R. E. Pratt,
and
V. J. Dzau.
Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells.
J. Clin. Invest.
83:
1419-1424,
1989.
29.
Newby, A. C.,
and
S. J. George.
Proposed roles for growth factors in mediating smooth muscle proliferation in vascular pathologies.
Cardiovasc. Res.
27:
1173-1183,
1993[Free Full Text].
30.
Ohtsubo, M.,
and
J. M. Roberts.
Cyclin-dependent regulation of G1 in mammalian fibroblasts.
Science
259:
1908-1912,
1993[Abstract/Free Full Text].
31.
Okada, K.,
C. Caramelo,
P. Tsai,
and
R. W. Schrier.
Effect of inhibition of Na+/K+-adenosine triphosphate on vascular action of vasopressin.
J. Clin. Invest.
86:
1241-1248,
1990.
32.
Okada, K.,
S. Ishikawa,
and
T. Saito.
Effect of a new V1 antagonist (OPC-21268) on vascular action of vasopressin in cultured rat vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
178:
707-712,
1991[Medline].
33.
Okada, K.,
S. Ishikawa,
and
T. Saito.
Mechanisms of vasopressin-induced increase in intracellular Na+ in vascular smooth muscle cells.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F1007-F1012,
1991[Abstract/Free Full Text].
34.
Okada, K.,
R. Sasaki,
S. Ishikawa,
and
T. Saito.
Distinct inhibition by non-peptide and peptide arginine vasopressin antagonists of vasopressin-induced activation of mitogen-activated protein kinase in cultured rat vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
200:
1155-1160,
1994[Medline].
35.
Onishi, T.,
W. Zhang,
X. Cao,
and
K. Hruska.
The mitogenic effect of parathyroid hormone is associated with E2F-dependent activation of cyclin-dependent kinase 1 (cdc2) in osteoblast precursors.
J. Bone Miner. Res.
12:
1596-605,
1997[Medline].
36.
Powell, J. S.,
J.-P. Clozel,
R. K. M. Mullar,
H. Kuhn,
F. Hefti,
M. Hosang,
and
H. R. Baumgartner.
Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury.
Science
245:
186-188,
1989[Abstract/Free Full Text].
37.
Sachinidis, A.,
Y. Ko,
W. Nettekoven,
A. J. Wieczorek,
R. Dusing,
and
H. Vetter.
The effect of angiotensin II on DNA synthesis varies considerably in vascular smooth muscle cells from different Wister-Kyoto rats.
J. Hypertens.
10:
1159-1164,
1992[Medline].
38.
Sadoshima, J.,
H. Aoki,
and
S. Izumo.
Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes.
Circ. Res.
80:
228-241,
1997[Abstract/Free Full Text].
39.
Schieffer, B.,
W. G. Paxton,
Q. Chai,
M. B. Marrero,
and
K. E. Bernstein.
Angiotensin II controls p21ras activity via pp60c-src.
J. Biol. Chem.
271:
10329-10333,
1996[Abstract/Free Full Text].
40.
Seed, B.,
and
J.-Y. Sheen.
A simple phase-extraction assay for chloramphenicol acyltransferase activity.
Gene
67:
271-277,
1988[Medline].
41.
Shimizu, M.,
E. Ichikawa,
U. Inoue,
T. Nakamura,
T. Nakajima,
H. Nojima,
H. Okayama,
and
K. Oda.
The G1/S boundary-specific enhancer of the rat cdc2 promoter.
Mol. Cell. Biol.
15:
2882-2892,
1995[Abstract].
42.
Spurr, N. K.,
A. C. Gough,
and
M. G. Lee.
Cloning of the mouse homologue of the yeast cell cycle control gene cdc2.
DNA Seq.
1:
49-54,
1990[Medline].
43.
Sugarman, J. L.,
A. H. Schonthal,
and
C. K. Glass.
Identification of a cell-type-specific and E2F-independent mechanism for repression of cdc2 transcription.
Mol. Cell. Biol.
15:
3282-3290,
1995[Abstract].
44.
Thatcher, J. D.,
B. McBride,
and
K. S. Katula.
Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.
DNA Cell Biol.
14:
869-881,
1995[Medline].
45.
Tommasi, S.,
and
G. P. Pfeifer.
In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression.
Mol. Cell. Biol.
15:
6901-6913,
1995[Abstract].
46.
Tsubota, Y.,
A. Takeuchi,
M. Shimizu,
T. Nakajima,
H. Nojima,
and
K. Oda.
Negative regulation of the rat cdc2 promoter in G1 by the silencer element.
Cell Growth Differ.
9:
257-265,
1998[Abstract].
47.
Watanabe, G.,
R. J. Lee,
C. Albanese,
W. E. Rainey,
D. Batlle,
and
R. G. Pestell.
Angiotensin II activation of cyclin D1-dependent kinase activity.
J. Biol. Chem.
271:
22570-22577,
1996[Abstract/Free Full Text].
48.
Watson, M. H.,
S. L. Venance,
S. C. Pang,
and
A. S. Mak.
Smooth muscle cell proliferation: expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.
Circ. Res.
73:
109-117,
1993[Abstract].
49.
Wen, S.-C.,
D.-H. Ku,
A. De Luca,
P. P. Claudio,
A. Giordano,
and
B. Calabretta.
Ets-2 regulates cdc2 kinase activity in mammalian cells: coordinated expression of cdc2 and cyclin A.
Exp. Cell Res.
217:
8-14,
1995[Medline].
50.
Yamamoto, M.,
M. Yoshida,
K. Ono,
T. Fujita,
N. Ohtani-Fujita,
T. Sakai,
and
T. Nikaido.
Effect of tumor suppressors on cell cycle-regulatory genes: RB suppresses p34cdc2 expression and normal p53 suppresses cyclin A expression.
Exp. Cell Res.
210:
94-101,
1994[Medline].
51.
Zwicker, J.,
F. C. Lucibello,
L. A. Wolfraim,
C. Gross,
M. Truss,
K. Engeland,
and
R. Müller.
Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanisms of transcriptional repression.
EMBO J.
14:
4514-4522,
1995[Medline].
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