(Received for publication, February 3, 1997, and in revised form, March 10, 1997)
From the Second Department of Internal Medicine, Osaka City University Medical School, Osaka 545, Japan and the § Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236, Japan
To elucidate the physiological role of protein
kinase C (PKC) , a ubiquitously expressed isoform in vascular smooth
muscle cells (VSMC), PKC
was stably overexpressed in A7r5 cells,
rat clonal VSMC. The [3H]thymidine incorporation in
A7r5 overexpressed with PKC
(DVs) was suppressed to 37.1 ± 16.3% (mean ± S.D.) of the level in control or A7r5 transfected
with vector alone (EVs). The reduction of [3H]thymidine
incorporation was strongly correlated with overexpressed PKC levels.
Moreover, transient transfection of a dominant negative mutant of PKC
restored the reduced proliferation in DVs. Flow cytometry analysis
demonstrated that DVs were arrested in the G0/G1 phase of the cell cycle. Expression of
cyclins D1 and E and retinoblastoma protein phosphorylation were
reduced, while the protein levels of p27 were elevated in DVs as
compared with EVs. There were no significant differences in the
expression of c-fos, c-jun, c-myc,
cyclin D2, D3, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and p21 among the clones. We
conclude that PKC
inhibits the proliferation of VSMC by arresting
cells in G1 via mainly inhibiting the expression of cyclin
D1 and cyclin E.
Proliferation of vascular smooth muscle cells (VSMC)1 plays a central role in the progression of atherosclerotic lesions (1, 2). In VSMC, activation of protein kinase C (PKC) has been shown to regulate cell differentiation and proliferation as well as modulate agonist-stimulated phospholipid turnover and increase the contractile force (3-5).
PKC is a complex family including three types of isozymes (6, 7). The
first group is the conventional PKCs (cPKC) (,
I,
II,
),
which are Ca2+-dependent and activated by
phorbol esters. The second is the novel PKCs (nPKC) (
,
,
,
, µ), which are also activated by phorbol esters but in a
Ca2+-independent manner. The third is the atypical PKCs
(
,
/
), which are activated in a
phosphatidylserine-dependent but Ca2+- or
phorbol ester-independent manner (4, 7, 8). The different PKC isozymes
are thought to have distinct functions regarding phosphorylation of
specific substrate proteins (7). Evidence accumulated to date suggests
that each PKC isozyme has a distinct role in cell proliferation. For
example, PKC
, a major isozyme ubiqitously expressed in most
mammalian cells, was reported to inhibit growth and induce
differentiation in fibroblasts (9, 10). PKC
was also reported to
inhibit proliferation and induce differentiation in melanoma cells
(11), while PKC
was shown to induce cell growth (10, 12). However,
little is known about the molecular mechanism by which individual PKC
isozymes control cell proliferation.
We found that PKC was expressed abundantly in rat aorta and rat
VSMC, but the physiological role of PKC
in VSMC and the signaling
events leading from it remains to be elucidated. In this study, we
first demonstrated the role of PKC
in VSMC growth and the cellular
mechanism involved. The cell cycle was arrested in the G1
phase in PKC
-overexpressing VSMC. The inhibition of cell
proliferation by PKC
was associated with suppression of G1 cyclin expression and retinoblastoma protein (pRb)
phosphorylation.
All dishes and plates were obtained from Becton Dickinson (San Jose, CA). All reagents, unless otherwise stated, were purchased from Sigma.
Cells and Cell CultureRat VSMC, A7r5, obtained from the American Type Culture Collection were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) (Life Technologies, Inc.) and used within eight passages. Before mitogenic stimulation, subconfluent cells were arrested in the quiescent state by a culture in DMEM containing 0.2% FCS for 72 h. Cell viability was greater than 95% by trypan blue exclusion for all experiments.
Generation of PKCFull-length PKC cDNA was cloned into the expression
vector SRD (13). A7r5 cells were transfected with either SRD vector alone as a control (designated as EVs) or with the PKC
expression vectors (designated as DVs) by the calcium phosphate precipitation method followed by 15% glycerol treatment as described previously (14). The transfected cells were subsequently grown in selection medium. SRD-based construct-transfected cells, being with the neomycin-resistant gene as a selection marker, were grown in DMEM containing 400 µg/ml G418. After 15-20 days in selection medium, single colonies were removed and subsequently examined for the expression of PKC protein by Western blot analysis.
Northern blot analysis was performed
as described previously (15). The probes used were as follows:
EcoRI fragments of mouse PKC cDNA (16), mouse PKC
I cDNA (16), mouse PKC
II cDNA (16), mouse PKC
cDNA (16), mouse PKC
cDNA (14), mouse PKC
cDNA
(13), mouse PKC
cDNA (17), mouse PKC
cDNA (18), cDNAs for human c-fos, c-jun, and
c-myc (obtained from the Japanese Cancer Research Resources
Bank), mouse cyclins D1, D2, D3, cyclin-dependent kinase
(cdk) 2 and cdk4 (kindly provided by Dr. H. Matsushime, University of
Tokyo, Tokyo, Japan), and EcoRI fragments of rat glyceraldehyde-3-phosphate dehydrogenase cDNA (19). Quantification of the relative signal intensities was performed with a densitometer using the software NIH Image (Bethesda, MD), and the results were expressed in arbitrary units.
The cultured VSMC were homogenized in ice-cold lysis buffer (20 mM Tris/HCl, pH 7.4, 300 mM sucrose, 2 mM EGTA, 5 mM EDTA, 0.3% (v/v) mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 5 mM 1,4-dithiothreitol, 600 µM diisopropyl fluorophosphate, and 25 µg/ml leupeptin). The homogenate was sonicated and then spun at 100,000 × g for 30 min, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in lysis buffer containing 1.0% Nonidet P-40 and agitated at 4 °C for 45 min. The homogenate was then sonicated and centrifuged at 100,000 × g for 30 min. The supernatant was used as the particulate fraction. Total cellular lysates were obtained by homogenizing cells in lysis buffer with 1.0% Nonidet P-40 and subjecting them to agitation for 45 min, followed by spinning at 100,000 × g for 30 min.
Western Blot AnalysisTotal cellular protein samples
(10-20 µg) were fractionated in 10% SDS-polyacrylamide gels and
electroblotted onto polyvinylidene fluoride membranes (Immobilon-P,
Millipore, Bedford, MA). The following highly specific polyclonal
antibodies were used for the analyses: antibodies directed against
peptides derived from ,
,
,
,
, and
subspecies of
PKC (Life Technologies, Inc.) and antibodies for cyclin D1, cyclin E,
p21, p27, pRb, cdk2, and cdk4 (Santa Cruz Biotechnology Inc., Santa
Cruz, CA). Western blots were developed by the ECL system (Amersham).
Specificities of the signals of PKC
and
were confirmed by using
other antibodies obtained from Santa Cruz Biotechnology Inc. The
densitometer was used to quantify the relative signal intensities of
the bands as described above.
[3H]Phorbol 12,13-dibutyrate (PDBu) binding was measured to estimate the levels of phorbol ester binding PKC molecule as described previously (14). Cells plated in a 48-well plate were washed twice with binding solution (DMEM, 1 mg/ml bovine serum albumin, 10 mM Hepes, pH 7.0) and incubated in the presence of 10 nM [3H]PDBu (DuPont NEN) at 37 °C for 30 min. Nonspecific binding was determined by incubating cells with [3H]PDBu in the presence of 10 µM unlabeled PDBu. Incubations were terminated by rapid washes with ice-cold phosphate-buffered saline and followed by a lysis with 0.1 N NaOH. Radioactivity in the lysates was determined by liquid scintillation counting.
Measurement of Ca2+-dependent and -independent Protein Kinase C ActivitiesCells were stimulated
with 100 nM 12-O-tetradecanoylphorbol-13-acetate
(TPA) for 15 min. The cytosolic fractions and Nonidet P-40-solubilized
particulate fractions obtained as stated above were applied to DE52
columns equilibrated with lysis buffer. PKC were eluted with lysis
buffer containing 0.1 M NaCl, and PKC activity in these
fractions was measured as described previously (14). The PKC assay
mixture containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 20 µM ATP, and 0.5 µCi of
[-32P]ATP (3000 Ci/mmol) (DuPont NEN) was incubated
with 50 µg/ml myelin basic protein4-14 for 15 min at
25 °C either in the presence of 0.5 mM Ca2+
and 25 µg/ml phosphatidylserine for cPKC activity or in the presence of 0.5 mM EGTA, 0.5 mM EDTA, and 25 µg/ml
cardiolipin for nPKC activity. The reaction was spotted onto DE52
paper, washed three times with 1% H3PO4, and
counted in a liquid scintillation counter.
A7r5 and transfected cells (EVs and DVs) were seeded in 24-well plates in DMEM with 10% FCS. Cell proliferation was determined by counting cell numbers. VSMC were trypsinized and counted every 24 h for 8 consecutive days using a hemocytometer. The average doubling time was calculated at a logarithmic growth phase. To determine the maximal cell density, cells grown in 24-well plates to confluency were kept for 4 additional days with daily medium changes and followed by cell counting. DNA synthesis was assessed by [3H]thymidine (DuPont NEN) incorporation as described previously with some modification (15). After 72 h of serum deprivation, cells in 48-well plates were stimulated with 10 ng/ml PDGF-BB (Genzyme, Cambridge, MA) for 24 h and labeled with [3H]thymidine for the last 2 h. Cells suspended by trypsinization were mixed vigorously with 5% (w/v) trichloroacetic acid and then centrifuged at 5,000 rpm for 3 min. Sediments were washed twice with 5% (w/v) trichloroacetic acid and three times with methanol. After the wash, the pellets were solubilized in formic acid, and the radioactivity was measured. The radioactivity was calibrated to the cell number of the well.
FACS AnalysesAfter 72 h of serum deprivation, control
and PKC -overexpressed cells were stimulated with 10 ng/ml PDGF. At
the indicated time, cells were suspended in trypsin EDTA, washed twice
with PBS, and fixed in 70% ethanol overnight at 4 °C. After washing once with PBS, fixed cells were incubated in 1 mg/ml RNase at 37 °C
for 30 min and followed by staining of the DNA with propidium iodide (1 µg/µl) at 4 °C for 2 h under shading. The cell cycle distribution was analyzed by FACScanTM (Becton Dickinson)
on the software "Cell FITTM DNA System" (Becton
Dickinson).
Dominant negative mutants of PKC (9) were expressed transiently in VSMC by
transferrin/polylysine-mediated gene delivery method (20) using a
"Transferrinfection Kit" (Bender Med Systems, Vienna, Austria). As
a control, empty vector alone was transfected. After seeding at 4 × 104 cells/well in a 48-well plate, cells were grown for
24 h in DMEM containing 10% FCS then incubated with 50 µM desferrioxamine at 37 °C for an additional 20 h. After the medium was changed, equal volumes of incubation mixture
containing 20 µM chloroquine and various concentrations
of transferrin/polylysine-conjugate·DNA complex were added. The basal
concentration (1 unit) of transferrin/polylysine-conjugate·DNA complex consisted of 25 µg/ml of expression or empty vector in Hepes-buffered saline (HBS; 20 mM Hepes, pH 7.3, 150 mM NaCl) and an equal volume of 25 units/ml
transferrin/polylysine in HBS (1 µg of DNA for 4 × 104 cells). After incubation with various
concentrations of mixture at 37 °C for 4 h, the cells were
washed, deprived of serum for 72 h, and analyzed for DNA
synthesis.
Statistical analysis was done by using analysis of variance combined with multiple comparison (Scheffe's type). These statistical analyses were carried out using Stat View IV on a personal computer (Macintosh Centris 650). The data were expressed as the mean ± S.D.
We
used A7r5, a clonal cell line of rat VSMC for the experiments. This
cell line has a monoclonal character and has been used as a model
system to examine proliferation and signal pathways including
mitogen-activated protein kinase, phospholipase D, and PKC (21, 22). We
examined the expression of PKC ,
I,
II,
,
,
,
,
and
in A7r5 by Northern and Western blot analyses and found that
PKC
and
were abundantly expressed (data not shown). PKC
,
,
, and
were also detectable (data not shown). To understand
the role of PKC
in VSMC and to elucidate the molecular signaling
event leading from PKC
, PKC
was stably transfected to A7r5.
Incorporation of vector and exogenous PKC
DNA into genomic DNA of
A7r5 was confirmed by Southern blot analysis (data not shown). DVs
showed 2.1-3.0- and 2.0-3.8-fold higher PKC
levels than A7r5 or
EVs as determined by Western blot and Northern blot analyses,
respectively (Fig. 1, A and B). No
differences in mRNA and protein levels of PKC
were observed
among DVs, A7r5, and EVs (Fig. 1, A and B).
PDBu binding activities, a simple method used to estimate the levels of
overexpressing PKC isozymes (23), in DVs were consistently 1.4-1.8-fold higher than in A7r5 or EVs (Fig. 1C). Although
PDBu binding assay measured more than just PKC , elevated PDBu
binding activities in DVs seemed to reflect the expression levels of
PKC
because no significant changes in mRNA or protein levels of PKC
, another abundantly expressed isoform, were appreciated. PKC
activities were also measured in EV6 and DV9, representatives of EVs
and DVs. cPKC activities in both soluble and particle fractions were
comparable between DV9 and EV6 (Table I). In contrast to cPKC, more than 50% of nPKC activity existed in the particle fraction even before cells were stimulated with TPA (Table I). The level of nPKC
activity in the particle fraction of DV9 was 2-fold higher than that of
EV6. There was no significant difference in nPKC activity between the
soluble fractions of DV9 and EV6. Expression of
smooth muscle
actin, a phenotypic marker for VSMC, in DV9 was as abundant as in EV6
as determined by Western blot analysis (data not shown).
|
DNA synthesis was
markedly suppressed in DVs compared with EVs 24 h after PDGF
stimulation (Fig. 2A). The level of
[3H]thymidine incorporation was negatively correlated
with that of PDBu binding (Fig. 2B) suggesting that DNA
synthesis was inversely associated with levels of PKC expression.
The time course of thymidine incorporation showed remarkable
suppression and delay of onset in DV9 compared with EV6 or A7r5 (Fig.
2C). DNA synthesis in A7r5 or EV6 started 12 h after
PDGF stimulation with the maximal accumulation observed at 24 h.
In contrast, it started in DV9 24 h after PDGF stimulation, and
the peak was observed at 48 h.
Cell numbers in the presence of 10% FCS for A7r5, EV6, and DV9 were counted every 24 h for 8 consecutive days (Fig. 2D). The proliferation of DV9 was significantly reduced as compared with A7r5 and EV6. Calculated doubling time of A7r5, EV6, and DV9 was 37.2 ± 0.8, 36.3 ± 4.8, and 62.7 ± 7.0 h, respectively. Moreover, the maximal cell density of DV9 was much lower than that of A7r5 or EV6 suggesting that DV9 cells were more susceptible to contact inhibition of growth (A7r5, 12.9 ± 0.35 × 104 cells/cm2; EV6, 13.0 ± 0.38 × 104 cells/cm2; DV9, 3.16 ± 0.45 × 104 cells/cm2).
To further elucidate the effect of PKC on cell growth, cell cycle
distribution was determined by flow cytometry. When EV6 cells were
deprived of serum for 72 h, 75.4 ± 4.77% of cells was arrested in the G0/G1 phase of the cell cycle.
When the cells were treated with PDGF for 24 h, 26.4 ± 7.32 and 15.6 ± 6.00% of EV6 cells were distributed in the S and
G2/M phase, respectively (Fig.
3A). Similar cell cycle distribution was
observed in A7r5 (data not shown). In contrast, 83.2 ± 2.28% of
DV9 cells were still in the G0/G1 phase 24 h after PDGF stimulation, and relatively few had progressed to the S
and G2/M phase 36 and 48 h after the stimulation (Fig.
3B). Thus, PKC
suppresses cell proliferation by
inhibiting and delaying G1/S transition in the cell
cycle.
To further elucidate the involvement of PKC in the regulation of
cell proliferation, we examined the effect of expression of a dominant
negative mutant of PKC
. This mutant harbors an amino acid
substitution at Lys-376 in the catalytic domain, which is crucial for
ATP binding and is conserved among several protein kinases (9).
Transfection of the dominant negative mutant of PKC
increased
PDGF-stimulated DNA synthesis in DV9 in a dose-dependent manner (Fig. 4B). In contrast, transfection
of the mutant did not affect PDGF-stimulated DNA synthesis in EV6 (Fig.
4A).
Suppression of G1 Cyclin Expression in PKC
We next examined the mechanism
underlying PKC suppression of cell proliferation. There was no
difference in the PDGF-stimulated increase in mRNA levels for
c-fos, c-jun, or c-myc among A7r5, EV6, and DV9 (Fig. 5). The maximal mRNA levels for
c-fos, c-jun, and c-myc after
stimulation with PDGF were observed at 30-45 min, 45-60 min, and
3 h, respectively, in each of the three cell lines (data not
shown).
Because PKC -overexpressing cells appear to be arrested in
G1 or delayed in G1/S transition, we examined
the expression of G1 cyclins and cdks necessary for
progression to S phase (24). Three types of cyclin D (D1, D2, and D3)
were detected in A7r5 by Northern blot analysis. In A7r5 or EV6, the
mRNA levels of cyclin D1 peaked at 6 h after PDGF stimulation,
while cyclin D2 and D3 were constitutively expressed and did not
oscillate (Fig. 6A). This result was in
agreement with previous reports (25, 26) showing the distinct
expression of three types of cyclin D underlying different regulation.
In marked contrast, a PDGF-stimulated increase in cyclin D1 mRNA
levels was only observed at 12 h with a maximal level at 18 h
in DV9 (Fig. 6A). 6 h after PDGF stimulation mRNA
levels of cyclin D1 in DV9 were significantly lower than those in EV6
(Fig. 6B).
Induction of cyclin D1 mRNA by PDGF was at least partly dependent on de novo protein synthesis, since treatment of cells with cycloheximide abolished PDGF-induced cyclin D1 mRNA at 18 h in both EV6 and DV9 (data not shown). Furthermore, treatment of cells with 100 µg/ml actinomycin D 6 h after PDGF stimulation showed that the half-life of cyclin D1 mRNA in DV9 was similar to that in EV6 (data not shown) suggesting that the stability of the mRNA for cyclin D1 was not altered in DV9.
Cyclin E mRNA levels were also increased by PDGF stimulation in EV6 (Fig. 6B). The basal level of cyclin E mRNA was significantly lower in DV9 than in EV6 cells, and PDGF did not increase the level in DV9 cells (Fig. 6B). There were no significant differences in mRNA levels of cyclin D2, D3, cdk2, and cdk4 between DV9 and EV6.
Immunoblot analysis showed that accumulation of cyclin D1 in EV6 was observed as early as 12 h after addition of PDGF with the maximal level at 18 h (data not shown). The peak level of cyclin E was observed later than that of cyclin D1 (24 h) (data not shown). On the other hand, cdk4 and cdk2 protein levels did not oscillate following PDGF stimulation (data not shown). In DV9, cyclin D1 and E protein levels were significantly lower than those in EV6 24 h after stimulation with PDGF, while cdk4 and cdk2 protein levels were comparable (Fig. 6C). Interestingly, basal and PDGF-regulated p27, an inhibitor of cdks (27, 28), levels were significantly higher in DV9 than in EV6 cells, while levels of p21, another cdk inhibitor (27, 28), were comparable among each cell line (Fig. 6C).
pRb phosphorylation, regulated by cyclin D- and E-associated cdks, is
necessary for cell cycle progression (24). In accordance with
down-regulation of cyclin D1 and cyclin E, pRb phosphorylation, which
was detected as a slower migrating band in immunoblot analysis, did not
occur for 24 h in DV9 (Fig. 6C). In contrast, in EV6
the hyperphosphorylated form of pRb was readily detected within 24 h after PDGF addition (Fig. 6C) and was observed for 48 h (data not shown). Thus, expression of cyclin D1 and cyclin E was
attenuated or delayed in PKC -overexpressing cells possibly
facilitating suppression of cell proliferation.
In this study, we first showed that PKC suppressed VSMC
proliferation, which was closely associated with inhibition of
G1/S transition and expression of G1 cyclins
(cyclin D1 and cyclin E).
We successfully
established a VSMC line in which PKC was overexpressed and found
that PKC
had a profound inhibitory effect on VSMC proliferation.
Using representative clones (DVs and EVs), we found that total PDBu
binding activities, which were up-regulated by PKC
transfection,
were inversely correlated to the levels of PDGF-stimulated DNA
synthesis. Furthermore, dominant negative PKC
compromised the
inhibitory effect of overexpressed PKC
on DNA synthesis. An
inhibitory role of PKC
in the cell proliferation was also reported
in fibroblast (6, 12). Taken together with these observations, PKC
appears to be an antimitogenic signaling molecule. However, the cell
cycle regulators governed by PKC
might be specific in cell species.
In VSMC overexpressed with PKC
, cell cycle was delayed or arrested
in the G1 phase, while in the Chinese hamster ovary cell,
Watanabe et al. (23) reported that PKC
arrested the cell
cycle at G2/M but not G1/S transition.
Expression of PKC is shown to be regulated by cell density in NIH
3T3 cells (29) and C6 glioma cells (30). Also in A7r5, PKC
mRNA
levels, but not PKC
, were significantly up-regulated by cell
confluency (data not shown) suggesting that PKC
is involved in
signaling from cell-cell interaction. This idea may be supported by the
observation that PKC
-overexpressing cells ceased growing at much
lower cell density than control cells. It will be important to
elucidate the physiological stimuli, including cell-cell interaction, and their signaling cascades leading to PKC
expression.
PKC has been shown to be
involved in PDGF-induced expression of the c-fos and
c-myc genes (31, 32). Hirai et al. (9) also
demonstrated that in NIH 3T3 cells PKC activates AP1/Jun transcription factor suggesting that these early responsive genes may
mediate the function of PKC
. However, we observed no changes in
mRNA levels of c-fos, c-jun, or
c-myc following PDGF stimulation in PKC
-overexpressing
VSMC, although we did not measure the transcriptional activity of
AP1/Jun or Myc/Max. Thus, PKC
may suppress the growth of VSMC
downstream of these early responsive genes.
We clearly demonstrated that phosphorylation of pRb and PDGF-stimulated
expression of cyclin D1 and cyclin E, but not cdk2, cdk4, cyclin D2, or
cyclin D3, were markedly suppressed in PKC -overexpressed VSMC.
Expression of cyclin D and E and phosphorylation of the pRb family are
indispensable for the progression of the G1 phase and for
entry into the S phase of the cell cycle in mammalian cells (33-35).
Cyclin D/cdk4 and cyclin E/cdk2 phosphorylate pRb during early-middle
G1 phase and late G1 phase, respectively (24). Although we did not measure cyclin D/cdk4 and cyclin E/cdk2 kinase activities, down-regulation of cyclin D1 and cyclin E proteins may
result in the abrogation of their associated kinase activities and pRb
phosphorylation. Cyclin/cdk activities are also inhibited by cdk
inhibitors such as p21 and p27 (27, 28). The protein level of p27, but
not p21, was elevated in PKC
-overexpressing VSMC. Thus, PKC
suppressed cyclin D1 and cyclin E and up-regulated p27, and this may
have led to abrogation of pRb phosphorylation and cell cycle
progression.
Phorbol ester has been shown to have a stimulatory (36), an inhibitory
(37), or no (38) effect on cyclin D1 expression in a cell
type-dependent manner. However, which PKC isozyme is involved in the regulation of cyclin D1 expression has yet to be
determined. We first directly demonstrated that PKC could suppress
cyclin D1 expression in VSMC. It is of interest to determine how PKC
regulates cyclin D1 expression in VSMC. In this study we showed
that the regulation of the level of cyclin D1 protein by PKC
was
associated with its mRNA level (Fig. 6). In addition, the half-life
of cyclin D1 mRNA was not affected by PKC
transfection, suggesting that mRNA levels for cyclin D1 are subjected to
transcriptional control by PKC
. We also observed that the
transcription of cyclin D1 was regulated through de novo
protein synthesis, which was in agreement with a previous report (39).
PKC
may regulate cyclin D1 transcription via this newly synthesized
protein, since treatment of EV6 and DV9 cells with cycloheximide
suppressed cyclin D1 mRNA induction by PDGF to the same level (data
not shown).
The mechanism of the suppression of cyclin E protein and mRNA by
PKC is not clear. It is known that cyclin E is regulated by a
transcription factor, E2F (40, 41), whose activity is controlled by
phosphorylation of pRb (34). Thus, abrogation of cyclin D1 expression
and pRb phosphorylation by PKC
may account for suppression of
cyclin E expression. However, since cyclin E expression is demonstrated
to be suppressed by TPA without affecting cyclin D1 expression (38), it
is also conceivable that PKC
suppresses the cyclin E expression
directly in a cell cycle-independent manner.
Recently specific PKC inhibitor was reported to ameliorate diabetic
vascular dysfunction (42). Clarifying specific biological functions of
distinct PKC isozymes in VSMC may provide us with a therapeutic
approach for preventing the progression of atherosclerotic disease.