Download Regulation of cdk2 Activity in Endothelial Cells That Are Inhibited

Regulation of cdk2 Activity in Endothelial Cells That Are
Inhibited From Growth by Cell Contact
Donghui Chen, Kenneth Walsh, Jian Wang
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Abstract—Endothelial cells (ECs) are quiescent in normal blood vessels but undergo rapid bursts of proliferation after
vascular injury and during angiogenesis. Here we show that the activity of cyclin-dependent kinase-2 (cdk2), a key
regulator of the G1 and S phases of the cell cycle, is expressed at high levels in proliferating ECs but at low levels in
ECs that are contact-inhibited for growth. Despite these differences in kinase activity, the protein levels of cdk2 and 1
of its activating subunits, cyclin E, are not modulated by these different growth conditions. The cdk inhibitor p27 is
highly expressed in contact-inhibited but not proliferating ECs, whereas the level of cyclin A protein is preferentially
expressed in proliferating ECs. p27 protein was detected in immunoprecipitable complexes with cdk2 or cyclin E in
cultures that were contact-inhibited for growth. The functional significance of the p27 induction was indicated by the
detection of a heat-stable cdk2 inhibitory activity that was induced by endothelial cell-cell contact and could be
immunodepleted with anti-p27 antibodies. In a confluent EC monolayer, cdk2 kinase activity was activated by a
scraping injury that led to cell migration and proliferation. The injury-induced activation of cdk2 coincided with the
downregulation of p27 and the induction of cyclin A. These data demonstrate that p27 is induced in confluent cultures
of ECs. They also indicate that both p27 induction and cyclin A downregulation contribute to the inhibition of cdk2 and
cell proliferation by cell-cell contact in ECs. (Arterioscler Thromb Vasc Biol. 2000;20:629-635.)
Key Words: cell cycle 䡲 contact inhibition 䡲 endothelium
U
nder normal conditions during adult life, the endothelia
of many organ systems remain in a quiescent, nonproliferative phenotype.1,2 The inhibition of endothelial cell (EC)
growth by cell-cell contact is in marked contrast to the rapid
burst of proliferation that occurs when the endothelial monolayer is disrupted, such as during abrasion of the vessel wall
by balloon angioplasty. High levels of EC proliferation are
also associated with microvascular morphogenesis, which
accompanies normal vascular development, and with the
abnormal ingrowth of new blood vessels that occurs during
tumor-induced angiogenesis.3,4 Despite the importance of EC
proliferation during pathogenesis and normal development,
little is known about the mechanisms that tightly couple
changes in cell cycle activity with changes in the EC
microenvironment.
Cell cycle progression is controlled by the periodic activation of cyclin-dependent kinases (cdks). cdks become activated by their association with activating subunits, referred to
as cyclins.5,6 The cdk4/cyclin D complexes function in the
early G1 phase of the cell cycle, whereas cdk2/cyclin E
complex is activated later in the G1 phase when the retinoblastoma gene product becomes phosphorylated. Previous
studies have demonstrated that cyclin E plays a key role in
regulating the transition from G1 to S. Overexpression of
cyclin E induces retinoblastoma protein hyperphosphoryla-
tion in an osteosarcoma cell line7 and shortens the G1 phase
in human fibroblasts.8,9 Microinjection of anti– cyclin E
antibody10 or anti-cdk2 antibody11 into human fibroblasts
prevents these cells from entering the S phase. Cyclin A,
another cdk2 partner, is upregulated when cyclin E levels are
downregulated. Expression of the cdk2/cyclin A holoenzyme
peaks in the S phase and is required for the elongation of
initiated replication and continuation of the S phase.12–14
cdk activity is also modulated through association with
negative regulatory subunits.15,16 A number of these cdk
inhibitors have been reported, including p21, p27, p16, p15,
p19, and p57.17–19 Two classes of cdk inhibitors have been
identified: those that are specific for cdk4 and/or cdk6 (p16,
p18, and p19) and those that have a broader specificity and
are referred to as general inhibitors (p21, p27, and p57).
These cdk inhibitors play a key role in cell growth inhibition
during cellular differentiation or in response to environmental
conditions that modulate cell growth, including exposure to
␥-irradiation, growth factors, and cytokines. Induction of the
cdk inhibitor p21 is associated with cell cycle withdrawal and
cell survival during differentiation of skeletal muscle,20,21
hematopoietic,22 neuronal,23 and hepatic24 cells. Induction of
the p27 cdk inhibitor has been observed in mink lung
epithelial cells that are exposed to transforming growth
factor-␤ or subjected to density-dependent growth inhibi-
Received January 1, 1999; revision accepted September 7, 1999.
From the Division of Cardiovascular Research (D.C., K.W., J.W.), St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Mass;
and the Cardiovascular Division (J.W.), Lilly Research Labs, Eli Lilly and Company, Indianapolis, Ind.
Correspondence to Jian Wang, PhD, Eli Lilly and Company, Mail Drop 0434, Lilly Corporate Center, Indianapolis, IN 46285. E-mail:
wang [email protected]
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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tion25,26 and in HeLa cells arrested in the G1 phase by
lovastatin.27 The expression of p27 has also been documented
in quiescent human T lymphocytes and is downregulated by
interleukin-2 treatment.28
EC proliferation is regulated by soluble growth factors,
such as vascular endothelial growth factor (VEGF) and
basic fibroblast growth factor (bFGF),1,29 as well insoluble
components of the extracellular matrix.30 It has been
shown that in vitro stimulation of EC proliferation by both
VEGF and bFGF requires activation of protein kinase
C.31,32 However, little is known about the regulation of cdk
complexes and activity in vascular ECs. Previously it has
been reported that the level of cyclin A mRNA is downregulated in ECs that are contact-inhibited for growth,33
but modulations in cyclin A protein level or their corresponding effects on cdk kinase activity were not evaluated.
Moreover, the role of p27 in controlling cdk activity in
ECs has not been elucidated. Here we show that cdk2 and
cyclin A– and cyclin E–associated kinase activities are
markedly reduced in contact-inhibited ECs, although levels of cdk2 protein are not affected by these different
growth conditions. We found that cyclin A protein levels
decline under conditions of cell-cell contact, but levels of
cyclin E do not change. In contrast, p27 protein and cdk
inhibitory activity are expressed in ECs that are contactinhibited for growth, and p27 is found in association with
the cdk/cyclin E complex in lysates prepared from these
cells. We also found that p27 is downregulated and cyclin
A upregulated in EC monolayers after a scraping injury
that induces cdk2 activity. These data suggest that alterations in p27 and cyclin A expression regulate EC proliferation during the transition from contact inhibition to the
proliferative state. This study represents the first systematic analysis of cdk activity and complex formation in
cell-cell contact–inhibited ECs.
Methods
Cell Culture
Bovine aorta ECs (BAECs) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS). Human umbilical vein ECs (HUVECs) were isolated
as described34 and grown in medium 199 (Life Technology) with
20% FBS, EC growth supplement (100 ␮g/mL), and heparin (50
U/mL). HUVECs between passages 1 and 5 were used in all
experiments. Human microvascular ECs (HMECs) of dermal origin
were purchased from Clonetics Corp (San Diego, Calif) and were
grow in EBM medium containing human epidermal growth factor
(10 ng/mL), hydrocortisone (10 mg/mL), 5% FBS, and 0.4% bovine
brain extract (Clonetics Corp). HMECs were used between passages
4 and 6. To achieve contact-inhibited cultures, cells were maintained
in growth medium for 3 more days after they reach confluence.
[3H]Thymidine Incorporation and Flow
Cytometry Analysis
Proliferating and contact-inhibited cultures of BAECs grown in
24-well plates were incubated with DMEM containing 300 ␮Ci/mL
[3H]thymidine (Dupont NEN) for 12 hours, washed with PBS, and
fixed in cold 10% trichloroacetic acid for 1 hour. The well was rinsed
with water, and trichloroacetic acid–precipitated material was solubilized with 0.25 mol/L NaOH. The tritium content of the NaOH
solution was determined by liquid scintillation counting in Scintiverse II (Fisher Scientific) with a Beckman LS 5000TD scintillation
counter. Parallel cultures of BAECs in 24-well plates were collected
by trypsinization, and cell numbers were determined by microscopy.
The [3H]thymidine radioactivity was expressed as counts per minutes
per 1000 cells. This thymidine incorporation rate was used as a
measure of DNA synthesis rate under the different culture
conditions.
For flow cytometry analysis, cells grown on Petri dishes were
collected with cell dissociation buffer (Life Technology), washed
with PBS, fixed in 70% ethanol overnight, and stained with propidium iodide (25 ␮g/mL, Sigma Chemical Co) in PBS with 1
␮g/mL RNase A. Flow cytometry analysis was carried out on a
Becton-Dickinson fluorescence-activated cell sorter.
Immunoblotting
Polyclonal antibodies against cdk2 (sc-163), cyclin A (sc-596),
cyclin E (sc-481), p21 (sc-397), p27 (sc-528), and p57 (sc-1040) and
the corresponding immunogenic peptide were all from Santa Cruz
Biotechnology. Cells lysates were prepared with NP-40 lysis buffer
(0.5% Nonidet P-40, 50 mmol/L Tris-HCI [pH 8.0], 250 mmol/L
NaCl, 2 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na2VO3,
1 mmol/L PMSF, and 2 ␮g/mL each of leupeptin and aprotinin) by
rocking at 4°C and cleared by centrifugation. For immunoblotting,
40 ␮g of cell lysates was separated on SDS–12% polyacrylamide
gels, transferred to nylon membranes (Millipore), blotted with the
indicated antibodies, and developed with the ECL chemiluminescence reagent (Amersham). For immunoprecipitation coupled with
immunoblotting, cell lysates (300 ␮g) were incubated with the
indicated antibodies and protein A–agarose beads (Boehringer
Mannheim), and the precipitated proteins were subjected
to immunoblotting.
cdk2 Kinase and Immunodepletion Assays
The cdk2-associated kinase assay and cdk2 inhibition assays were all
performed as described.35 For cdk2 kinase assay, cell lysates (100
␮g) were immunoprecipitated with anti-cdk2 antibody. The precipitated protein complexes were incubated with histone H1 (100
␮g/mL) and [␥-32P]ATP (5 ␮Ci) in kinase buffer (50 mmol/L Tris
[pH 8.0], 10 mmol/L MgCl2) for 30 minutes at room temperature.
The reactions were terminated by the addition of SDS–loading buffer
and separated on SDS gel exposed to x-ray film to detect histone H1
incorporation of [␥-32P]ATP.
For the kinase inhibition assay, 400 ␮g of cell lysates was boiled
for 5 minutes to inactivate endogenous cdk2 activity. These heattreated lysates were mixed with cdk2 immunoprecipitates from
proliferating BAEC lysates (100 ␮g) for 1 hour at room temperature
and subjected to the kinase assay. For immunodepletion, the heattreated lysates were precleared by immunoprecipitation with antip27 antibody in the presence or absence of p27 immunogenic peptide
before being mixed with the cdk2 immunoprecipitates.
Injury of the EC Monolayer
Injury to EC monolayers was performed by a protocol described
previously.36 In brief, contact-inhibited BAEC monolayers grown on
100-mm plates were scraped with a stainless steel rake, which
uniformly swept out ⬇50% of the cells. Wounded BAEC monolayers were washed to remove floating cells and cellular debris and
incubated in fresh medium to induce cell migration and proliferation.
Cells were collected at different time points after injury and
subjected to immunoblotting analysis.
Results
Regulation of cdk2 Kinase Activity and Cell Cycle
Protein Levels in ECs
We first measured the DNA synthesis rates in subconfluent
versus confluent BAECs under the culture conditions that
were employed for the subsequent cell cycle analyses. Rates
of [3H]thymidine incorporation were 40-fold higher in subconfluent cultures of BAECs than in confluent cultures on a
per-cell basis despite the presence of 10% FBS in the medium
(Figure 1A). The cell cycle distribution of BAECs in subconfluent and confluent cultures was also determined by flow
Chen et al
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Figure 1. A, [3H]Thymidine incorporation in BAECs. Subconfluent and confluent cultures of BAECs were incubated with medium containing [3H]thymidine for 12 hours, and [3H]thymidine incorporation rates were measured. Cell numbers from parallel cultures of BAECs
treated under the same conditions were determined, and the [3H]thymidine incorporation rate was expressed on the histogram relative
to cell number. B, Cell cycle distribution of subconfluent and confluent BAECs. The x axis indicates the level of propidium iodide fluorescence, which corresponds to the DNA content per cell. The y axis indicates the relative frequency of events. This experiment was
repeated twice, and similar results were obtained.
cytometry analysis. In cultures of subconfluent BAECs, 58%
of the cells were in the G0/G1 phases of the cell cycle,
whereas 24% were in the S and 17% in the G2/M phases. In
contrast, in confluent cultures of BAECs, only 13% of the
cells were in the G2/M⫹S phases of the cell cycle (Figure
1B). These data, consistent with a previous report,37 demonstrate that under conditions of confluence, BAECs display
cell-cell contact growth inhibition that arrests cells in the
G0/G1 phases of the cell cycle.
To investigate the molecular basis of cell-cell contact
growth inhibition in ECs, the levels of the cdk2-associated
kinase activities were measured in extracts prepared from
subconfluent and confluent cultures of BAECs. Immunoprecipitates of cdk2, cyclin E, or cyclin A prepared from
proliferating cultures of BAECs all exhibited high levels of
histone H1 kinase activity, whereas immunoprecipitates prepared from confluent cultures uniformly displayed low levels
of kinase activity (Figure 2). Thus, the levels of cdk2 activity
were correlated with the proliferative activities displayed by
BAEC cultures under these different growth conditions.
The expression levels of cdk2, cyclin A, and cyclin E
proteins were analyzed in BAECs. Immunoblot analyses
revealed that the levels of cdk2 and cyclin E were similar
between cultures of proliferating BAECs and that cultures of
BAECs had been contact-inhibited for growth (Figure 3A). In
contrast, cyclin A was expressed at much lower levels in the
contact-inhibited cells. This expression pattern of cyclin A
protein parallels that of its transcript under these different
growth conditions. A low level of cyclin A protein is likely to
contribute to the reduced cdk2/cyclin A kinase activity that is
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Figure 2. Downregulation of cdk2-associated kinase activity in
BAECs that were contact-inhibited for growth. Cell lysates from
proliferating (P) or confluent (C) cultures of BAECs were immunoprecipitated with anti-cdk2, anti– cyclin (cyc) A, or anti– cyclin
E antibodies. The immunoprecipitates were incubated with
[32P]ATP and histone H1. The reaction mix was separated on
SDS–polyacrylamide gel electrophoresis gels, and the relative
kinase activity of the immunoprecipitates is indicated by histone
H1 incorporation of [32P]ATP. The relative kinase activities
detected in lysates of confluent cultures are presented in the
histogram as percentages of the kinase activity in lysates of
proliferating BAECs. Mean values and SDs were calculated from
3 independent experiments.
observed in contact-inhibited BAECs (Figure 2). However,
differences in cdk2/cyclin E kinase activity between proliferating and growth-arrested cultures are likely to be controlled by a different mechanism, since there was no corresponding change in the level of cyclin E.
Immunoblots analyzing the cdk inhibitor p27 revealed that
its expression in ECs was influenced by cell-cell contact. A
low level of p27 was detected in proliferating cultures of
subconfluent BAECs. However, p27 was expressed at markedly higher levels in BAEC cultures that were contactinhibited for growth (Figure 3A). To investigate whether p27
upregulation is a common feature of cell contact–mediated
growth inhibition in ECs, immunoblot analyses were per-
Figure 3. Immunoblotting analysis of cyclins (Cyc), cdk2, and
p27 in proliferating versus confluent ECs. A, Cell lysates (40 ␮g)
from proliferating BAECs (P) or BAECs that were inhibited for
growth by cell-cell contact (C) were subjected to immunoblotting analysis with the indicated cyclin, cdk2, and p27 antibodies.
The experiment was repeated 3 times, and a similar result was
obtained. On average, levels of cdk2 and cyclin E were similar
between proliferating and contact-inhibited BAECs. The levels of
cyclin A in contact-inhibited BAECs decreased by a factor of
3.2, whereas the levels of p27 increased 4.3-fold. B, Protein levels of cdk2 and p27 in proliferating (P) versus confluent (C)
HUVECs were determined by immunoblot. The experiment was
repeated 3 times, and a similar result was obtained. On average, levels of cdk2 were similar between proliferating and
contact-inhibited BAECs. The levels of p27 increased 2.7-fold.
C, Immunoblotting of p27 in proliferating and confluent HMECs.
The experiment was repeated 3 times, and a similar result was
obtained. On average, levels of cdk2 were similar between proliferating and contact-inhibited BAECs. The levels of p27
increased 2.4-fold.
Figure 4. Immunoprecipitation coupled with immunoblotting
revealed an association of p27 with cdk2 and cyclin E in
contact-inhibited BAECs. A, Cell lysates from proliferating (P) or
contact-inhibited (C) BAECs were immunoprecipitated with anti–
cyclin E antibody in the absence (⫺) or presence (⫹) of immunogenic peptide for cyclin E. The immunoprecipitates were
loaded on an SDS–polyacrylamide gel electrophoresis gel and
immunoblotted with anti-cdk2, anti-p27, and anti– cyclin E antibodies sequentially. Both cdk2 and p27 were present in immunoprecipitable cyclin E complex from contact-inhibited BAEC
lysates. In contrast, only cdk2 was present in the cyclin E complex from proliferating BAEC lysates. B, cdk2 immunoprecipitates from proliferating or contact-inhibited BAEC lysates were
subjected to immunoblotting with anti-p27 and anti-cdk2 antibodies sequentially. cdk2 was associated with p27 in contactinhibited but not proliferating BAECs. These experiments were
repeated 3 times with similar results.
formed with cell lysates prepared from HUVECs. As shown
in Figure 3B, p27 is expressed at low levels in proliferating
cultures of subconfluent HUVECs and at substantially higher
levels in cultures of HUVECs that were contact-inhibited for
growth. In contrast, these different culture conditions did not
influence the expression of cdk2 protein. Furthermore,
HMECs also expressed higher levels of p27 under conditions
of contact-inhibited growth arrest than in proliferating cultures (Figure 3C). Thus, high levels of p27 expression appear
to be a common feature in ECs that are growth-arrested due
to cell-cell contact. High levels of p27 in cell-cell contact–
inhibited ECs may be responsible for the low levels of
cdk2/cyclin E activity and partially responsible for the low
cdk2/cyclin A activity in these cells.
Potential negative regulators of cdk2 activity also include
p21 and p57. Using an antibody that can detect p21 from a
broad range of species, we were not able to detect significant
levels of p21 in BAECs or HUVECs by Western blot
analysis. This result is consistent with a previous report that
p21 was not detected in ECs and smooth muscle cells from
normal porcine iliofemoral arteries.37a Using an antibody
specific for human p57, we were not able to detect significant
levels of p57 in HUVECs. Thus, in contrast to findings in
muscle and other cell types,22,38 Western blot analyses failed
to detect significant levels of p21 or p57 expression (data not
shown) in ECs.
p27 Is Bound to cdk2/Cyclin E Complexes in
Contact-Inhibited ECs
Immunoprecipitation-coupled immunoblotting analyses were
performed to determine the proteins associated with p27 in
BAEC extracts (Figure 4A). The cyclin E–associated proteins
were immunoprecipitated with anti– cyclin E antibody and
subsequently subjected to immunoblotting with anti-cdk2 and
Chen et al
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Figure 5. p27 is a component of the cdk2 inhibitory activity
detected in contact-inhibited but not proliferating BAECs. Heattreated cell lysates (400 ␮g) from proliferating (P) or contactinhibited (C) BAECs were incubated with cdk2 immunoprecipitates from proliferating BAECs for 1 hour before the cdk2 kinase
assay. A heat-stable cdk2 inhibitory activity was detected in
contact-inhibited but not proliferating BAEC lysates (lanes 2 and
4). Preclearance by immunoprecipitation with anti-p27 antibody
largely depleted this cdk2 inhibitory activity (lane 5). The presence of p27 immunogenic peptide prevented immunodepletion
of this cdk2 inhibitory activity (lane 6). The mean values from 3
independent experiments are expressed in the histogram with
standard error bars. Film exposure from 1 representative experiment is shown.
anti-p27 antibodies. cdk2, but not p27, was detected in cyclin
E immunoprecipitates from lysates of proliferating BAECs.
In contrast, both cdk2 and p27 were detected in cyclin E
immunoprecipitates from lysates of contact-inhibited BAECs.
Similarly, p27 could be detected in cdk2 immunoprecipitates
from lysates of contact-inhibited BAECs but not from lysates
of proliferating BAECs (Figure 4B).
cdk2 Inhibitory Activity in Contact-Inhibited ECs
Is Immunodepleted With Anti-p27 Antibodies
A number of cdk inhibitors are small, heat-stable molecules.
Thus, heat-treated lysates can be used to analyze cdk inhibitor
activities,35 in the absence of interfering cdks that are heatlabile. Heat-treated lysates from proliferating and contactinhibited BAECs were assayed for their cdk inhibitory
activity (Figure 5). A heat-stable cdk2 inhibitory activity was
detected in contact-inhibited BAEC lysates, but not in proliferating BAEC lysates. The large portion of this cdk2
inhibitory activity was immunodepleted by pretreatment with
an anti-p27 antibody. An excess of immunogenic p27 peptide
prevented the anti-p27 antibody from removing the cdk2
inhibitory activity, demonstrating the specificity of the immunodepletion reaction. Collectively, these data indicate that
p27 contributes significantly to the inhibition of cdk2 activity
in BAECs that are contact-inhibited for growth.
p27 Downregulation Is Correlated With EC
Migration and Proliferation in Response to a
Scraping Injury
To measure the cell density– dependent expression level of cdk2,
cyclin A, cyclin E, and p27, we implemented an endothelial
cdk Regulation in ECs by Cell Contact
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Figure 6. Effect of a scraping injury on cdk2 activity and cell
cycle protein expression in EC monolayers. Confluent BAEC
monolayers were injured with a steel rake (see Methods) and
incubated in medium contain 10% FBS to induce cell migration
and proliferation (see Methods). Cell lysates were collected
before injury (C); at the time of injury (0); at 15 and 30 minutes
after injury; and at 1, 2, 4, and 6 hours after injury. Extracts
were prepared and assayed for cdk2 activity and for the expression of p27, cdk2, cyclin A, and cyclin E by immunoblotting with
appropriate antibodies. The relative levels of p27, cyclin A,
cyclin E, and cdk2 were also measured with a densitometer and
expressed on the histogram (upper panel). The experiment was
performed multiple times with similar results.
wound-healing model and examined the steady-state levels of
these proteins during the cellular response to injury, which
occurs as cell-cell contacts are disrupted and the induction of cell
migration ensues. With this model system, a uniform population
response to injury is achieved by using a specially designed rake,
which sweeps out 400- to 600-␮m-wide zones of cells in
concentric arrays, leaving an endothelial population that is
migrating and proliferating (in unison) during the response to
injury. Cell lysates were prepared from BAEC monolayers at
different time points after the scraping injury and assayed for
cdk2 activity and the protein levels of p27, cyclin A, cyclin E,
and cdk2 (Figure 6). The scraping injury led to a marked
induction of cdk2 activity by 4 hours. High levels of cdk2
activity were maintained at 6 and 8 hours after injury, but it was
diminished by 10 hours and later time points. The levels of cdk2
protein and cyclin E did not change significantly over this time
course. On the other hand, the upregulation of cdk2 activity at 4
hours coincided with the maximal decrease in p27 levels (a
factor of 4) and the maximal increase in cyclin A levels
(⬇2.5-fold). By 14 hours after injury, the decrease in cdk2
activity coincided with an increase in p27 level and a reduction
in cyclin A level. Immunohistochemical analysis of bromode-
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March 2000
oxyuridine incorporation revealed active DNA synthesis in the
forefront of migrating cells between 8 and 14 hours after injury
but not at later time points (data not shown). These data
demonstrate a good correlation between the induction of DNA
synthesis by a scraping injury and changes in p27 and cyclin A
levels. Furthermore, these data suggest that the coordinate
regulation of p27 and cyclin A may have a role in controlling EC
proliferation during wound healing.
Discussion
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cdk2 performs key regulatory roles in the G1 and S phases of
the cell cycle. Here we have demonstrated a marked reduction
of cdk2 kinase activity in ECs that are contact-inhibited for
growth compared with cells that are proliferating. To determine the regulatory mechanisms that contribute to this
reduction in cdk2 activity, we analyzed the expression and
protein interactions of cdk2 and its regulatory subunits in EC
cultures under these different growth conditions. Initial measurements revealed similar levels of cdk2 protein in proliferating and growth-arrested ECs, suggesting that differential
interactions with cdk2-associated protein might play a role in
cdk2 regulation under these conditions. The experiments
described herein demonstrate that at least 2 mechanisms
contribute to the reduction in cdk2 activity in ECs that are
contact-inhibited for growth.
The cdk2/cyclin E holoenzyme functions in the G1 phase of
the cell cycle. In view of the important role of cyclin E in early
cell cycle control, it is interesting that this peptide is not
downregulated in ECs when they are contact-inhibited for
growth and that immunoprecipitable cdk2/cyclin E complexes
can be detected in both proliferating and growth-arrested ECs.
The observation that lysates prepared from contact-inhibited
ECs do not exhibit appreciable cyclin E–associated kinase
activity suggested that inhibitory subunits might be involved in
the regulation of cdk2 under these conditions. Our data show
that p27 expression was markedly enhanced in confluent EC
cultures that were contact-inhibited for growth. These data are
consistent with a previous report that an elevated p27 protein
level is associated with cell-cell contact or transforming growth
factor-␤ treatment–induced growth arrest.39
Immunoprecipitation-coupled immunoblot analyses were
performed on EC extracts to test whether the binding of p27
to cdk2 might be responsible for the reduction in cdk2 kinase
activity in ECs that were contact-inhibited for growth. Lysates prepared from confluent cultures of growth-arrested
ECs demonstrated interactions between p27 and cdk2, p27
and cyclin E, and cyclin E and cdk2. Furthermore, lysates
prepared from growth-arrested but not proliferating ECs
displayed a heat-stable cdk2 inhibitory activity that could be
immunodepleted by pretreatment with anti-p27 antibodies.
Collectively, these data indicate that the cdk2/cyclin E
holoenzyme, whose expression is not repressed by cell-cell
contact in ECs, is inactivated by the association of p27
inhibitory subunits under conditions of cell contact–mediated
growth inhibition. Furthermore, the p27 level is rapidly and
transiently downregulated after a scraping injury to EC
monolayers, which induces transient EC proliferation and
migration. These data suggest that p27 plays a key role in
maintaining the growth-inhibitory status of ECs under condition of cell-cell contact and that its downregulation is
involved for these cells to reenter the cell cycle.
We have also show here that cyclin A protein levels were
substantially reduced in the confluent cultures of the growtharrested ECs. This finding is consistent with a previous report
showing that cyclin A mRNA level and promoter activity were
lower in confluent cultures of ECs than in subconfluent cultures
of ECs.33 We demonstrated that EC lysates contained reduced
levels of cyclin A–associated kinase activity under conditions
that lead to cell contact–induced growth arrest. Since the levels
of the cdk2/cyclin A holoenzyme peak during the S phase and it
is required for both the initiation and elongation of DNA
replication,12–14 reductions in the level of this cyclin are likely to
contribute to the growth arrest of ECs by inhibiting the cell cycle
machinery that functions during the S phase of the EC cycle. In
support of this hypothesis, we found that cyclin A was induced
by a scraping injury to EC monolayers and that its induction was
maintained for up to 14 hours.
Recently, it was reported that p27 can block cyclin A
transcription in fibroblasts40 and vascular smooth muscle
cells.41 This inhibitory effect of p27 requires the E2F binding
site located at position ⫺37 to ⫺32 in the cyclin A promoter.40 Increased levels of p27 can inhibit cdk2/cyclin E activity
and release p107 and the retinoblastoma tumor suppressor
protein that, in turn, can inhibit E2F activity. Thus, it is
plausible that an elevated level of p27 is instrumental in the
repression of cyclin A expression in ECs under cell-cell
contact conditions. However, this may not be the only
pathway, since the cAMP response element– biding site at
position ⫺79 to ⫺72 in the cyclin A promoter has also been
shown to be required for the repression of cyclin A transcription in confluent ECs.33
Interestingly, it has been reported that expression of the p21
cdk inhibitor can be regulated by the extracellular matrix
through a p53-dependent pathway in ECs. Agonists of EC
integrin ␣V␤3 have been shown to suppress p53 transcription
activity, inhibit p21 expression, and increase the bcl-2/bax level,
which promotes EC survival.42 Conversely, ␣V␤3 antagonists
activate p53 and increase p21 expression, leading to EC apoptosis.43 Therefore, it appears that p27 is involved in cell-cell
contact–mediated EC growth inhibition, whereas p21 is involved
in EC survival mediated by the extracellular matrix.
Protein kinase Cs are involved in both VEGF- and bFGFmediated EC proliferation.31,32 Recent reports indicate that
protein kinase Cs exert their effects on EC proliferation via
regulation of the cell cycle machinery, at least in part. It has
also been shown that phorbol ester and diacylglycerol inhibit
cdk2 activity and the G2/M transition in ECs through the
suppression of cdc25B expression.44 Harrington et al45 reported that forced expression of protein kinase C␦ in ECs
resulted in delayed passage through the S phase of the cell
cycle. Collectively, these reports suggest that a diverse array
of cell cycle proteins is involved in regulating EC proliferation in response to different environmental conditions.
In summary, we have shown here that cdk2 activity is low in
ECs that are inhibited for growth by cell-cell contact. Two
mechanisms can account for this reduction in cdk2 activity:
(1) a reduction in cyclin A and (2) the upregulation of p27,
which binds and inactivates the cdk2/cyclin E holoenzyme. The
integrity of the endothelial monolayer is a key component in the
development of atherosclerotic lesions,46 and the acceleration of
endothelial healing after arterial injury can inhibit the formation
of restenotic lesions.47 Therefore, further studies on EC cycle
Chen et al
regulation may have important implications for understanding
the basic mechanisms of atherogenesis and postinterventional
restenosis.
Acknowledgments
This work was supported by National Institutes of Health grants HL
50692, AG 15052, and AR 40197 to K. Walsh. We thank L. Truong
and K. Shook for technical assistance.
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Regulation of cdk2 Activity in Endothelial Cells That Are Inhibited From Growth by Cell
Contact
Donghui Chen, Kenneth Walsh and Jian Wang
Arterioscler Thromb Vasc Biol. 2000;20:629-635
doi: 10.1161/01.ATV.20.3.629
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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