© 2000 American Heart Association, Inc.
Vascular Biology |
Regulation of cdk2 Activity in Endothelial Cells That Are Inhibited From Growth by Cell Contact
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_jian{at}lilly.com
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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.
Key Words: cell cycle • contact inhibition • endothelium
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Introduction |
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Under 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 hyperphosphorylation 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 13 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 18 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-ß or
subjected to density-dependent growth inhibition25 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 contact-inhibited 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.
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Methods |
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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 µg/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 µCi/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 µg/mL, Sigma Chemical Co) in PBS with 1 µg/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 µg/mL each of leupeptin and aprotinin) by rocking at 4°C and
cleared by centrifugation. For
immunoblotting, 40 µg 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 µg) 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 µg) were immunoprecipitated with anti-cdk2 antibody. The
precipitated protein complexes were incubated with histone H1 (100
µg/mL) and [-32P]ATP (5 µCi) 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 µg of cell lysates was boiled for 5 minutes to inactivate endogenous cdk2 activity. These heat-treated lysates were mixed with cdk2 immunoprecipitates from proliferating BAEC lysates (100 µg) for 1 hour at room temperature and subjected to the kinase assay. For immunodepletion, the heat-treated lysates were precleared by immunoprecipitation with anti-p27 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.
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Results |
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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
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.
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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.
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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 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.
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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 contact-inhibited for growth (Figure 3A). To investigate whether p27 upregulation is a common feature
of cell contact–mediated growth inhibition in ECs,
immunoblot analyses were performed 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 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).
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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 heat-labile. Heat-treated lysates from
proliferating and contact-inhibited 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.
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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 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-µm-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 bromodeoxyuridine 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.
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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-ß 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 growth-arrested 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 13 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
Vß3 have been shown to suppress p53 transcription activity,
inhibit p21 expression, and increase the bcl-2/bax level, which
promotes EC survival.42 Conversely, Vß3
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 bFGF-mediated 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 regulation may have important implications for understanding the basic mechanisms of atherogenesis and postinterventional restenosis.
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Acknowledgments |
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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.
Received January 1, 1999; accepted September 7, 1999.
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