Division of Nephrology, Department of Medicine, Department of Veterans Affairs Medical Center and University of Iowa College of Medicine, Iowa City, Iowa 52242-1081
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ABSTRACT |
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The mechanism by which the bradykinin B1 receptor (B1R) inhibits platelet-derived growth factor (PDGF)-stimulated proliferation was investigated in cultured rat mesenteric arterial smooth muscle cells. The B1R agonist des-Arg9-bradykinin (DABK) was found to inhibit PDGF-mediated activation of the cyclin E-cyclin-dependent kinase 2 (Cdk2) complex and to prevent hyperphosphorylation of retinoblastoma protein. DABK did not inhibit upregulation of cyclin E expression but increased expression of the Cdk2 inhibitor p27Kip1 and the association of p27Kip1 with the cyclin E-Cdk2 complex. In addition, DABK inhibited the PDGF-stimulated expression of cyclin D that would otherwise siphon p27Kip1 away from inhibition of cyclin E-Cdk2. The signaling mechanism by which DABK regulated p27Kip1 was explored. DABK was found to stimulate the activity of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) and to prolong activation of MEK and ERK by PDGF. Inhibition of ERK activation with the MEK inhibitors PD-98059 and U-0126 as well as the Src family kinase inhibitor PP2 completely blocked the effect of DABK to increase p27Kip1 and partially reversed the DABK-mediated inhibition of PDGF-stimulated proliferation. These studies demonstrate that the B1R inhibits PDGF-stimulated mitogenesis in part by prolonged activation of ERK leading to increased expression of p27Kip1.
vascular smooth muscle; cell division; cyclin-dependent kinases; cyclins; mitogen-activated protein kinases; Src family kinases; signal transduction
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INTRODUCTION |
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THE BRADYKININ B1 receptor (B1R) is a cytokine-inducible G protein-coupled receptor that is upregulated on vascular smooth muscle cells after vascular injury (25, 31). It has also been localized on cells within human atheromatous plaque (33). The physiological role of the B1R is not known. We previously investigated (11) the effects of the B1R in cultured vascular smooth muscle cells and found that activation of the B1R inhibits growth factor-stimulated mitogenesis. Recent studies have further shown that activation of the B1R inhibits neointimal formation after vascular injury in vivo (1, 14). These studies suggest that the B1R is upregulated by vascular injury and can stimulate signaling pathways that inhibit vascular smooth muscle cell proliferation.
The mechanism by which activation of the B1R inhibits smooth muscle cell proliferation has not been elucidated. Our previous study (11) demonstrated that stimulation of the B1R acted late in G1 phase to block platelet-derived growth factor (PDGF)-induced cell cycle progression into S phase (DNA synthesis). The present study was performed to examine the cellular mechanism whereby des-Arg9-bradykinin (DABK) inhibits cell cycle progression in late G1. Potent growth factors such as PDGF stimulate progression from G1 into S phase by stimulating the sequential upregulation of the G1-phase regulatory proteins cyclin D and cyclin E, which bind and activate cyclin-dependant kinases (Cdk) (37). Three different isoforms of cyclin D (D1, D2, and D3) bind and activate either Cdk4 or Cdk6, whereas cyclin E and cyclin A activate Cdk2 (37). The sequential phosphorylation of retinoblastoma protein (Rb) by cyclin D-Cdk4/6 and cyclin E-Cdk2 inactivates Rb and releases the transcription factor E2F from Rb-mediated repression, leading to increased transcription of important S-phase genes (37). Inhibition of G1 cyclin-Cdk activity may be mediated by inhibition of cyclin expression, alterations in the phosphorylation state of Cdk, or increases in Cdk inhibitory proteins such as p27Kip1 or p21Cip1 (28, 38). The present study examined the hypothesis that activation of the B1R inhibits mitogenesis by blocking the activation of cyclin E-Cdk2 in the late G1 phase of the cell cycle. Further studies were also performed to explore the mechanism of this inhibition.
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EXPERIMENTAL PROCEDURES |
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Chemicals.
DABK was obtained from Bachem (Torrance, CA); recombinant human
PDGF-BB, recombinant human interleukin (IL)-1,
2'-amino-3'-methoxyflavone (PD-98059),
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U-0126), and
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) were obtained from Calbiochem (San Diego, CA). Antibodies were
purchased from the following sources. A monoclonal antibody against Rb
was from BD PharMingen (Lexington, KY). Antibodies against the
phosphorylated active forms of p42/44 extracellular signal-regulated
kinase (ERK) (T202/Y204), active phosphorylated mitogen-activated
protein kinase kinase (MEK) (S217/S222), as well as total p42 ERK,
total MEK, and horseradish peroxidase (HRP)-conjugated secondary
antibodies were from Cell Signaling Technology (Beverly, MA). All other
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Protein A-Sepharose CL4B was from Amersham Pharmacia Biotech
(Piscataway, NJ). Radionuclides [
-32P]ATP (10 Ci/mmol)
and [3H]thymidine (10-20 Ci/mmol) were from
DuPont-NEN (Boston, MA). SuperSignal West Femto, Dura, and Pico
chemiluminescence reagents were from Pierce (Rockford, IL). All other
chemicals were purchased from Sigma (St. Louis, MO). Immobilon-P was
obtained from Millipore (Bedford, MA).
Cell culture. Arterial smooth muscle cells were isolated from the mesenteric artery of 5-wk-old male Sprague-Dawley rats from Harlan (Indianapolis, IN) and grown in culture as previously described (10). Cultures were grown in Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% bovine calf serum under an atmosphere of 5% CO2-95% air at 37°C until the cultures attained confluence (4-7 days). To arrest the cells in G1, the cells were incubated in defined serum-free medium (50:50 mixture of Ham's F-12 and DMEM supplemented with 5 mg/l insulin, 5 mg/l transferrin, 5 nM triiodothyroxine, 10 nM sodium selenite, 100 U/ml penicillin, and 100 µg/ml streptomycin) for 4 days before study. Only the first and second passages were used for these studies.
The B1R is upregulated by cytokines such as IL-1Immunoprecipitation kinase assay.
After treatment with the experimental agents as described in
RESULTS, cells were washed twice with ice-cold
phosphate-buffered saline (PBS) and extracted into ice-cold lysis
buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 2.5 mM EGTA, 1 mM NaF, 0.1 mM
Na3VO4, 10 mM -glycerophosphate, 1 mM
dithiothreitol, 0.1% Tween, 10% glycerol, 10 µg/ml leupeptin,
containing fresh 0.91 trypsin inhibitor units/ml aprotinin and 100 µg/ml PMSF; 4 ml/10-cm dish). All steps were done at 4°C.
The samples were precleared with nonimmune rabbit IgG (1 µg/ 500 µl) and protein A-Sepharose. Immunoprecipitation of Cdk2, cyclin E,
or cyclin A was performed with 1 µg of the appropriate polyclonal
antibody per 500 µl. After incubating for 8-12 h at 4°C the
immune complexes were precipitated with protein A-Sepharose and washed
three times with 500 µl of lysis buffer followed by two washes with
kinase buffer (10 mM HEPES, pH 7.5, 2.5 mM EGTA, 1 mM dithiothreitol,
10 mM MgCl2, 0.1 mM Na3VO4, 1 mM
NaF, and 10 mM
-glycerophosphate). The final pellet was resuspended
in a final volume of 20 µl of kinase buffer, and the kinase reaction
was started by adding 10 µl of reaction buffer (5 µg histone H1, 1 nmol ATP, 3 µCi [
-32P]ATP in kinase buffer). The
samples were incubated for 30 min at 30°C before the reaction was
stopped by adding 20 µl of 3× SDS sample buffer and boiling for 5 min. The resulting supernatants were resolved by SDS-PAGE (12% gel)
and electroblotted to Immobilon-P followed by autoradiography to detect
the phosphorylated histone.
Immunoblotting. The procedures followed those previously described (12). After treatment with experimental agents as described in RESULTS, cells were washed twice with ice-cold PBS and extracted into ice-cold lysis buffer as described in Immunoprecipitation kinase assay. The samples were analyzed with SDS-PAGE (7.5-15% gel as appropriate) followed by electroblotting (Owl Separation Systems) onto Immobilon-P. Incubation with the primary antibody (0.5-1 µg/ml) and horseradish peroxidase-conjugated secondary antibody was performed in Blotto, and the antibody-labeled proteins were detected by enhanced chemiluminescence with Kodak X-OMAT XAR film (Rochester, NY). The resulting autoradiogram was analyzed by densitometry with a scanner (ScanMaker4, Microtek Lab) to capture the image into Microsoft Photoeditor. The resulting electronic image file was analyzed by 1-D image analysis software (Kodak Digital Science, Rochester, NY) to quantitate the intensity of each signal.
[3H]thymidine incorporation.
The methods followed those previously reported (11).
Briefly, cells were plated in 48-well panels at an initial density of
3.5 × 104 per well and allowed to proliferate in DMEM
containing 10% fetal calf serum. When the cultures attained confluence
the medium was replaced with defined serum-free medium (see Cell
culture) for 4 days to arrest the cells in
G0/G1. The cells were pretreated with IL-1
(2 ng/ml) for 12-24 h in fresh serum-free medium before addition
of the additives outlined in RESULTS.
[3H]thymidine (0.5 µCi/well) was added to the wells
immediately after addition of the experimental agents (1 h after adding
PDGF), and the cells were allowed to incubate at 37°C under an
atmosphere of 5% CO2-95% air for a total of 32 h
after addition of PDGF. The cells were then washed sequentially with
ice-cold PBS (0.5 ml × 2), 10% trichloroacetic acid (0.5 ml × 1) and 95% ethanol (0.5 ml × 1) before the DNA was extracted
into 0.5 ml of 1 N NaOH followed by 0.5 ml of 1% SDS. The extracts
were combined, and [3H]thymidine incorporation was
quantitated by liquid scintillation counting (Beckman LS3801).
Statistics.
Data are expressed as means ± SE. Comparisons were performed by
nonparametric statistics using Friedman's two-way ANOVA for multiple
comparisons followed by the Wilcoxon signed rank test to analyze for
significant differences between individual groups. Statistics are
reported for the primary comparison of interest, the effect of
PDGF + DABK compared with PDGF alone. A P 0.05 was considered statistically significant.
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RESULTS |
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DABK inhibits PDGF-stimulated cyclin E- and cyclin A-Cdk2 activity.
Our previous studies (11) showed that DABK inhibits
PDGF-stimulated mitogenesis in the late G1 phase of the
cell cycle just preceding entry into S phase. Activation of cyclin
E-Cdk2 and subsequently cyclin A-Cdk2 is required for cell cycle
progression in this phase of the cell cycle. We therefore examined the
effect of DABK on the PDGF-stimulated activity of these enzyme
complexes with immunoprecipitation kinase assays. As shown in Fig.
1, treatment of serum-deprived arterial
smooth muscle cells with PDGF produced a time-dependent increase in the
activity of total Cdk2 and Cdk2 activity immunoprecipitated with cyclin
E and cyclin A. Activation of Cdk2 produces hyperphosphorylation of its
endogenous target Rb. The hyperphosphorylation can be detected by the
presence of a slower-mobility band on SDS gel electrophoresis
(13), and this was also observed after treatment with PDGF
(Fig. 2A). Treatment with the
B1R agonist DABK alone had no effect on cyclin-dependent activation of
Cdk2 or phosphorylation of Rb (not shown). However, addition of DABK
after PDGF completely inhibited the PDGF-stimulated cyclin-dependent
activation of Cdk2 (Fig. 1) and hyperphosphorylation of Rb (Fig. 2).
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Effect of DABK on PDGF-stimulated G1-phase cell cycle proteins. Cdk2 is constitutively present in cells and depends on an increase in cyclin E and cyclin A for its activation. As shown in Fig. 2, treatment with PDGF increased the expression of cyclin E and subsequently cyclin A. Cdk2 was present in quiescent cells but was further increased by PDGF. Treatment with PDGF also produced a higher-mobility Cdk2 band (see Fig. 2A, bottom) consistent with the presence of an activating phosphorylation of Cdk2 at T160 (30). Addition of DABK did not prevent the PDGF-stimulated expression of cyclin E or the increase in Cdk2 but completely blocked the increase in cyclin A and the activating phosphorylation of Cdk2. Because upregulation of cyclin A is dependent on activation of cyclin E-Cdk2 (45), the results indicated that inhibition by DABK occurred at the level of cyclin E-Cdk2 but did not depend on inhibition of cyclin E expression.
An increase in Cdk2 inhibitory proteins p21Cip1 or p27Kip1 can produce inactivation of cyclin E-Cdk2 and impaired Cdk2 phosphorylation on T160 (30). We examined this possibility (Fig. 2) and found that PDGF produced an increase in p21Cip1 but this was not further increased by treatment with DABK, indicating that p21Cip1 did not mediate the inhibitory effect of DABK. Conversely, p27Kip1 was typically expressed at a high level in quiescent, confluent cells and tended to decrease after treatment with PDGF. As shown in Fig. 2, addition of DABK reversed the PDGF-mediated inhibition and produced an increase in p27Kip1 expression that peaked at 12 h. The timing of this increase in p27Kip1 occurred when cells would normally activate cyclin E-Cdk2 (Fig. 1), hyperphosphorylate Rb (Fig. 2), and pass from G1 into S phase (11). A related Cdk2 inhibitory protein, p57Kip2, was also present in vascular smooth muscle cells but did not change after treatment with PDGF or DABK (data not shown). These results are consistent with a role for p27Kip1 in mediation of the antiproliferative effects of DABK. The effectiveness of p27Kip1 to inhibit cyclin-Cdk2 activity also depends on the mitogen-induced expression of cyclin D and formation of cyclin D-Cdk4/6 (7). These cyclin D-Cdk4/6 complexes bind p27Kip1 but are resistant to its inhibition (7). By binding p27Kip1, cyclin D-Cdk4/6 complexes serve to decrease inhibition of cyclin E-Cdk2 (8, 29). As shown in Fig. 2, PDGF stimulated a time-dependent increase in the expression of cyclins D1-3 as well as an increase in Cdk4 and Cdk6. Addition of DABK inhibited the PDGF-induced expression of all three early G1 cyclins, cyclin D1, D2, and D3 as well as producing a delayed inhibition of Cdk4 but not Cdk6. The inhibition of cyclin D2 and D3 by DABK occurred early and preceded or coincided with the peak in p27Kip1 at 12 h (Fig. 2). Conversely, the inhibition of cyclin D1 and Cdk4 did not become significant until after 12 h, when the increase in p27Kip1 and inhibition of cell cycle progression had already occurred (Fig. 2). These results suggest that inhibition of PDGF-induced mitogenesis by DABK may depend on both increased expression of p27Kip1 and decreased expression of cyclin D2 and D3.DABK enhances p27Kip1 binding to cyclin E-Cdk2.
We next examined whether treatment with DABK increased the
amount of p27Kip1 bound to cyclin E-Cdk2. As shown in
Fig. 3, p27Kip1 was present in quiescent
cells and was decreased by treatment with PDGF for 12 h. Addition
of DABK reversed the PDGF-stimulated decrease in p27Kip1. Cyclin E was
immunoprecipitated, and the associated Cdk2 activity as well as the
coimmunoprecipitated p27Kip1 was measured. PDGF stimulated an increase
in cyclin E expression and the associated cyclin E-Cdk2 activity.
However, the amount of p27Kip1 that immunoprecipitated with cyclin E
was unchanged compared with basal conditions, indicating that there was
less p27Kip1 bound for each molecule of cyclin E. Conversely, treatment with DABK inhibited the PDGF-stimulated increase in cyclin E-Cdk2 activity, and this was associated with an increase in p27Kip1 bound to
cyclin E-Cdk2.
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DABK prolongs PDGF activation of MEK and ERK.
The signal transduction pathway that mediates the antiproliferative
effect of the B1R is unknown (11). Activation of p42/p44 ERK has been reported to upregulate expression of cyclin D and inhibit
the expression of p27Kip1 (20, 24, 34). We therefore explored whether DABK would inhibit PDGF-stimulated activation of ERK.
As shown in Fig. 4, at early time points,
DABK alone produced a rapid activation of ERK that was qualitatively
similar but quantitatively less than that produced by PDGF. Combined
treatment with DABK and PDGF did not inhibit the effect of PDGF at
early time points but instead prolonged the overall duration of
activation of ERK by PDGF. We examined this directly by looking at
later time points that corresponded to those we used for cell cycle
analysis. These studies demonstrated that, compared with PDGF, combined
treatment with PDGF + DABK produced a significant two- to
threefold increase in ERK activity and nearly an eightfold increase in
MEK activity at 4 h (Fig. 4). The difference between the two
treatments persisted for at least 8 h. Hence, DABK did not inhibit
but prolonged the PDGF-stimulated activation of MEK and ERK.
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Effect of MEK inhibition on cyclin D and p27Kip1 expression.
Whereas activation of ERK would not explain the effect of DABK to
decrease cyclin D expression, there are conflicting data regarding the
effect of p42/44 ERK on regulation of p27Kip1 (18, 20, 34, 42,
44). We therefore explored whether the prolonged activation of
ERK by DABK altered the PDGF-mediated expression of p27Kip1 by using
two different inhibitors of MEK, PD-98059 and U-0126. These inhibitors
block the activity of MEK1/2 responsible for phosphorylation and
activation of ERK (4, 16). For these experiments, the
inhibitors were added 30 min after stimulation with PDGF but 30 min
before addition of DABK, and the expression of p27Kip1 was measured at
12 h after addition of PDGF. When given before the agonist,
pretreatment with either of these inhibitors blocked both PDGF- and
DABK-stimulated activation of ERK at 30 min (not shown). When the
inhibitors were given 30 min after PDGF but before DABK they inhibited
the enhanced activation of ERK produced by the combination of DABK with
PDGF at 4 h (Fig. 5). In addition,
the overall level of ERK activity in both quiescent and PDGF-stimulated
cells was decreased compared with vehicle-treated controls (Fig. 5) . As expected, U-0126 was more effective than PD-98059 at inhibiting the
DABK-mediated augmentation of PDGF-stimulated ERK activity at 4 h
(Fig. 5). As shown in Fig.
6A,
treatment with PD-98059 and U-0126 completely blocked
the ability of DABK to increase p27Kip1 [percent increase in p27Kip1
stimulated by DABK + PDGF compared with PDGF alone: control
154 ± 41.5% (P < 0.05), U-0126 44.3 ± 14.1%, PD-98059 28.5 ± 17.7%]. There was also a tendency for
these inhibitors to enhance the inhibitory effect of PDGF on p27Kip1
expression [percent inhibition by PDGF compared with basal: control
21.8 ± 13.4%, U-0126 38.4 ± 8.4%, PD-98059 67.5 ± 5.8% (P < 0.05 compared with control)]. These
results imply that in primary cultures of arterial smooth muscle cells
the PDGF-mediated activation of ERK may serve to partially
counterbalance other PDGF-stimulated signals that inhibit p27Kip1
expression. Addition of DABK further prolongs ERK activation and
overrides these other PDGF-stimulated inhibitory signals to produce an
increase in p27Kip1 expression. The MEK inhibitors were also found to
increase the basal expression of p27Kip1 in serum-deprived quiescent
cells (Fig. 6A). This may suggest that the low levels of
activated ERK in quiescent cells have a different effect on p27Kip1
expression compared with mitogen-stimulated cells.
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Effect of a SFK inhibitor. Activation of a SFK has been shown to be involved in the downstream activation of ERK by many G protein-coupled receptors (9, 19). As shown in Fig. 5, treatment with the SFK inhibitor PP2 inhibited PDGF- and DABK-mediated activation of ERK. In contrast to the MEK inhibitors, PP2 did not inhibit basal levels of active ERK in quiescent cells (Fig. 5). We further explored the effect of PP2 on the DABK-mediated increase in p27Kip1 and decrease in cyclin D. Similar to the effect of the MEK inhibitors, treatment with PP2 completely blocked the ability of DABK to increase p27Kip1 expression but did not prevent DABK from inhibiting expression of cyclin D2 or cyclin D3. Of interest, PP2 was also found to inhibit the PDGF-induced expression of cyclin D1 and D3, suggesting a role for a SFK in the PDGF-stimulated expression of these cyclins. Together, these results are consistent with the notion that DABK acts via a SFK to prolong PDGF activation of ERK, leading to increased expression of p27Kip1 but not the inhibition of cyclin D by DABK.
Inhibitors of MEK and SFK partially reverse antiproliferative
effect of DABK.
Because both MEK and SFK inhibitors blocked the DABK-mediated
upregulation of p27Kip1, we further examined whether they would also
prevent the DABK-mediated inhibition of PDGF-stimulated mitogenesis. As
shown in Fig. 7, compared with the
vehicle-treated control, treatment with PD-98059 and U-0126 inhibited
basal thymidine incorporation. Comparison with Fig. 6 and Table
1 suggests that the inhibition of basal
activity was associated with an increase in basal levels of p27Kip1
expression. Treatment with any of the three inhibitors (PD-98059,
U-0126, or PP2) also partially inhibited PDGF-stimulated mitogenesis.
In the absence of inhibitors, addition of DABK 1 h after exposure
to PDGF inhibited PDGF-stimulated mitogenesis in these experiments by
56.8 ± 2.2% (Fig. 7). Addition of PD-98059, U-0126, or PP2 30 min before addition of DABK partially reversed the inhibitory effect of
DABK [DABK-mediated inhibition of PDGF-stimulated mitogenesis was
19.5 ± 6.1%, 18.3 ± 2.4%, and 40 ± 2.9% after treatment with PD-98059, U-0126, and PP2, respectively
(P < 0.001 for each compared with control)]. These
results imply that activation of ERK and upregulation of p27Kip1 play a
role in mediating the antiproliferative effects of DABK. However, DABK
must also activate other pathways such as those leading to inhibition
of cyclin D expression that contribute to the inhibition of
PDGF-stimulated mitogenesis.
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Effect of DABK on cell cycle proteins and thymidine incorporation in absence of PDGF. As shown above, DABK can modestly activate ERK in the absence of PDGF. Moreover, previous studies reported that DABK alone can stimulate proliferation of quiescent vascular smooth muscle cells from rabbit aorta (25). Hence, DABK might have effects on cell cycle regulatory proteins in the absence of PDGF. As shown in Table 1, treatment with DABK for 12 h produced a modest increase in the expression of cyclin D1. However, DABK did not increase [3H]thymidine incorporation (Table 1), activate cyclin E-Cdk2 (not shown), or significantly alter the expression of cyclin D2 or D3 or p27Kip1 (Table 1). We also examined the effect of the MEK and SFK inhibitors when DABK was given alone. As demonstrated above, the MEK inhibitors increased basal expression of all the measured cell cycle proteins and decreased basal thymidine incorporation. Treatment with DABK was found to inhibit p27Kip1 expression in the presence of U-0126 but not PD-98059. Otherwise, the MEK inhibitors did not significantly alter the effects of DABK seen in vehicle-treated control cells. In these studies, PP2 also increased basal levels of these cell cycle proteins. In the presence of PP2, DABK was found to inhibit expression of p27Kip1 and cyclin D3 and to produce a modest inhibition of basal thymidine incorporation. These data demonstrate that DABK was not a mitogen in these cells. Moreover, the effect of DABK to increase expression of p27Kip1 was only seen in the presence of combined treatment with PDGF (compare Fig. 2 and Table 1).
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DISCUSSION |
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Kinins acting via B1 and B2 receptors produce inhibition of growth factor-stimulated arterial smooth muscle proliferation in vitro and can inhibit neointimal formation after vascular injury in vivo (1, 11, 15). The present study demonstrates that activation of the B1R inhibits arterial smooth muscle cell proliferation by inhibiting the activity of cyclin E-Cdk2 required for transit through the late G1 to S phase of the cell cycle.
Growth factors such as PDGF activate cyclin E-Cdk2 by stimulating an increase in cyclin E that binds to the constitutively expressed Cdk2. Activation of cyclin E-Cdk2 further requires phosphorylation of Cdk2 on T160 in the activation loop by cyclin-activating kinase (CAK) (28). Treatment with DABK did not inhibit the PDGF-induced expression of cyclin E but produced an inactive cyclin E-Cdk2 complex that lacked the gel mobility shift characteristic of the activating phosphorylation on Cdk2. One established mechanism by which hormones can inhibit the cyclin E-Cdk2 complex is upregulation of one of a family of Cdk2 inhibitory proteins such as p21Cip1 or p27Kip1 (30, 38). Binding of p21Cip1 or p27Kip1 to the cyclin-Cdk2 complex disrupts the structure of Cdk2, thereby preventing phosphorylation of T160 and activation of the complex. We found that DABK did not alter the effect of PDGF on p21Cip1 or p57Kip2. However, DABK did reverse the PDGF-mediated decrease in p27Kip1 and produced a marked increase in p27Kip1 as well as in the amount of p27Kip1 complexed with cyclin E. The timing of this effect was appropriate, as the upregulation of p27Kip1 expression occurred at 12 h, just when cyclin E-Cdk2 becomes active (Fig. 1), Rb becomes hyperphosphorylated (Fig. 2), and cells are just entering S phase (11). Finally, inhibitors of MEK and SFK that prevent the DABK-mediated upregulation of p27Kip1 partially reversed the inhibitory effects of DABK on PDGF-stimulated proliferation. Together, these results are consistent with the hypothesis that p27Kip1 participates in the inhibitory effect of DABK on cyclin E-Cdk2. However, the results also demonstrate that additional mechanisms must exist for DABK to fully inhibit PDGF-stimulated mitogenesis.
The ability of p27Kip1 to inhibit cyclin E-Cdk2 also depends on the level of expression of cyclin D (8, 41). Mitogens such as PDGF act in the early G1 phase of the cell cycle to stimulate cyclin D and its activation of Cdk4 or Cdk6. However, binding of p27Kip1 and/or p21Cip1 is required to promote formation and nuclear translocation of the active cyclin D-Cdk4/6 complex (7, 23). Consequently, mitogenic upregulation of cyclin D serves to siphon p27Kip1 into a complex with cyclin D-Cdk4/6 and away from inhibition of cyclin E-Cdk2 (8, 30, 41). The resulting activation of cyclin E-Cdk2 can phosphorylate p27Kip1 on T187 leading to ubiquitinylation and proteosome-mediated degradation of p27Kip1 (27). In the present study, DABK was found to inhibit PDGF-stimulated upregulation of all three cyclin D isoforms as well as the expression of Cdk4 but not Cdk6. In light of the timing of the effect, inhibition of cyclins D2 and D3 is likely to be most important for inhibiting progression into S phase (Fig. 2). By inhibiting the expression of cyclin D and Cdk4, DABK not only inhibits the cyclin D-Cdk4/6-mediated phosphorylation of Rb but also reduces the number of cyclin D-Cdk4/6 complexes that are available to bind p27Kip1. Hence, p27Kip1 remains bound to cyclin E-Cdk2 and inhibits its activity. Together, these observations suggest that the decrease in cyclin D also plays a major role in the antiproliferative effect of DABK.
Prior studies from our laboratory (11) demonstrated that prostaglandins do not mediate the antiproliferative effect of DABK, and the signal transduction pathway by which the B1 receptor inhibits PDGF-stimulated mitogenesis in arterial smooth muscle cells is unknown. PDGF activates Ras and triggers the MEK/ERK cascade that can promote cell cycle progression by upregulating cyclin D (24, 39). Consistent with these prior observations, we found that inhibition of MEK with either PD-98059 or U-0126 partially inhibited the PDGF-mediated increase in cyclin D and stimulation of mitogenesis. However, treatment with DABK was found to activate, rather than inhibit, ERK. Moreover, cotreatment with DABK prolonged the PDGF-mediated activation of both MEK and ERK. Hence, DABK does not inhibit cyclin D expression by inhibiting the PDGF-stimulated activation of ERK. Moreover, the prolonged activation of ERK by combined treatment with DABK and PDGF did not prevent DABK from inhibiting the PDGF-stimulated expression of cyclin D. Together these results indicate that DABK acts via an alternate pathway to inhibit expression of cyclin D.
In contrast to the effect on cyclin D expression, we found that inhibition of MEK completely blocked the ability of DABK to increase p27Kip1 when combined with PDGF. Interestingly, in the absence of combined treatment, isolated treatment with either PDGF or DABK tended to decrease expression of p27Kip1, and this effect was variably enhanced by inhibition of MEK. These observations suggest that activation of ERK, particularly when it is prolonged by combined treatment with DABK and PDGF, stimulates increased expression of p27Kip1. Moreover, inhibition of MEK may further unmask alternative PDGF- and possibly DABK-stimulated signaling pathways that inhibit p27Kip1 expression. The mechanism whereby MEK inhibitors increased expression of p27Kip1 in unstimulated, quiescent cells is unclear. Given the low level of active ERK in these quiescent cells, the effect of the MEK inhibitors to increase p27Kip1 may not be mediated by inhibition of MEK. Alternatively, these results may suggest that inhibition of the basal MEK activity in quiescent cells has an opposite effect on expression of p27Kip1 (as well as cyclin D) compared with mitogen-stimulated arterial smooth muscle cells. This latter hypothesis suggests that the effect of inhibiting ERK on p27Kip1 and cyclin D expression may depend on cell context (i.e., early G0/G1 phase vs. late G1/S phase). Further experiments will be needed to sort out these possibilities. Nevertheless, in PDGF-stimulated cells the prolonged activation of ERK appears to mediate the increase in p27Kip1 expression produced by DABK and to counterbalance other PDGF-activated signals that inhibit expression of p27Kip1.
Previous studies have examined the effect of Ras and the Raf/MEK/ERK cascade on p27Kip1 expression in various cell lines and have found conflicting results (8, 18, 20, 21, 34, 39, 42, 44). Generally, activation of Ras by mitogens leads to a decrease in p27Kip1 expression (2, 3, 20, 21, 34, 39). However, the Ras-mediated inhibition of p27Kip1 expression may involve signaling pathways other than the Raf/MEK/ERK cascade, such as phosphatidylinositol-3-kinase (PI3K) (6, 26). Evidence for the role of the MEK/ERK pathway in mediating the effect of Ras to inhibit Kip1 expression is controversial (6, 8, 18, 20, 34, 39, 42). Studies using expression of activated forms of Raf have reported a decrease in p27Kip1 expression that can be reversed by inhibition of MEK (20, 34). However, studies using activated forms of MEK have not consistently found inhibition of p27Kip1 expression (8, 18). Moreover, studies examining the effect of hormones on cell cycle progression suggest a more complex role for MEK/ERK activation to regulate p27Kip1 expression (39, 42). Similar to the present report, Wolf and Stahl (42) found that angiotensin II (ANG II) produced an increase in p27Kip1 expression that was prevented by MEK inhibitors. In addition, Weber et al. (39) showed that PDGF stimulated a decrease in p27Kip1 expression, but this was dependent on activation of PI3K and not the MEK/ERK cascade. These prior studies were performed in various cell lines that typically lack one or more constraints regulating cell cycle progression. Hence, some of these cell lines may have genetic alterations that effect how signaling pathways regulate p27Kip1 and inhibit cell cycle progression. From the results of the present study, when primary cultures of arterial smooth muscle cells are stimulated to reenter the cell cycle by PDGF the increase in p27Kip1 by DABK is dependent on activation of ERK.
One question raised by these results is how prolongation of PDGF-stimulated ERK activity by DABK can lead to increased p27Kip1 expression and contribute to inhibition of PDGF-stimulated mitogenesis. In this regard, studies by Woods et al. (43) using transfection of an inducible form of Raf are illuminating. Woods et al. (43) showed that expression of p21Cip1 and activation of mitogenesis depend on the level of activation of Raf and ERK. Modest levels of Raf activation stimulated mitogenesis, but higher levels of Raf activation led to an increase in p21Cip1 expression and inhibition of mitogenesis. Hence, the strength of activation of the Raf/MEK/ERK cascade can determine the effect on p21Cip1 expression and mitogenesis. The present study suggests that the level of ERK activation may also regulate the expression of p27Kip1 in vascular smooth muscle cells. Further studies will be needed to fully understand how signals stimulated by DABK and PDGF combine to prolong activation of ERK and how this activation integrates with other signaling pathways to regulate expression of p27Kip1 and p21Cip1 in these cells.
A second question raised by this study is why activation of ERK, which is regarded as a mitogenic signaling pathway (22, 35), would increase a Cdk2 inhibitor. As discussed above, p27Kip1 or p21Cip1 can bind to the cyclin D-Cdk4/6 complex and promote activation and nuclear localization of the complex (7, 23). Hence, activation of the Raf/MEK/ERK cascade may promote increased expression of p27Kip1 or p21Cip1 to assist in the activation and nuclear translocation of cyclin D-Cdk4/6. This concept is consistent with studies discussed above showing that activation of ERK upregulates p21Cip1 (32, 43).
For some G protein-coupled receptors the activation of Ras and the Raf/MEK/ERK cascade has been found to involve a SFK (9). In the present study, the SFK inhibitor PP2 was found to prevent DABK-mediated activation of ERK and to block upregulation of p27Kip1 when DABK was given with PDGF. Moreover, treatment with PP2 also led to a partial reversal of the DABK-mediated inhibition of PDGF-stimulated mitogenesis. Collectively, these data indicate that DABK increases p27Kip1 expression in vascular smooth muscle cells by activating a SFK and the MEK/ERK cascade.
Activation of ERK has been reported to be necessary but not sufficient
to stimulate mitogenesis (40). Moreover, agonists such as
ANG II that stimulate ERK lead to hypertrophy rather than proliferation
(17, 42). Inhibition of p27Kip1 with antisense oligonucleotides has been shown to block the hypertrophy and increase the proliferative response to ANG II (5, 42). Thus the
hypertrophy produced by ANG II appears to be due to the sustained
expression of p27Kip1 that prevents the ANG II-mediated increase in
cyclin D from stimulating DNA synthesis and subsequent cell division (5, 36). Collectively, these observations and the results of the present study suggest the model shown in Fig.
8. In arterial smooth muscle cells,
activation of ERK by growth factors such as ANG II stimulates an
increase in cyclin D and p27Kip1 that promotes hypertrophy rather than
proliferation. Potent mitogens such as PDGF stimulate proliferation
because they activate additional pathways (e.g., PI3K or Rho GTPase)
that override the effect of ERK and inhibit expression of p27Kip1
(6, 26). Alternatively, DABK inhibits PDGF-induced
proliferation by activating an alternate pathway that inhibits
expression of cyclin D while promoting the ERK-induced expression of
p27Kip1. Although both DABK and ANG II increase p27Kip1, ANG II does
not inhibit PDGF-stimulated mitogenesis because, in contrast to DABK,
it does not inhibit expression of cyclin D. These effects of ERK
activation are likely to depend on cell context (e.g., timing in the
cell cycle) and the strength or duration of ERK activation. When viewed
in this light, selective activation of ERK may be primarily a
hypertrophic rather than a proliferative pathway in vascular smooth
muscle cells.
|
In summary, the results of the present study now provide a molecular framework to understand the mechanism whereby activation of the B1R can inhibit vascular smooth muscle cell proliferation and prevent neointimal proliferation after vascular injury. Future studies will need to elucidate how activation of the B1R prolongs the PDGF-induced activation of ERK and how this leads to increased expression of p27Kip1. In addition, the alternate pathway by which DABK inhibits PDGF-stimulated expression of cyclin D remains to be determined. Elucidating these pathways may open up new therapeutic approaches to regulate neointimal proliferation after vascular injury.
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ACKNOWLEDGEMENTS |
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This research was supported by a Department of Veterans Affairs Merit Review award, a grant-in-aid from the American Heart Association, the University of Iowa Diabetes and Endocrinology Research Center, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25295.
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FOOTNOTES |
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Portions of this work were previously presented at the American Society of Nephrology Meetings, Toronto, Ontario, Canada, October 2000, and published in abstract form (J Am Soc Nephrol 11: 453A, 2000).
Address for reprint requests and other correspondence: B. S. Dixon, Div. of Nephrology, Dept. of Medicine, Dept. Veterans Affairs Medical Center and Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081 (E-mail: bradley-dixon{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 6, 2002;10.1152/ajpcell.00289.2001
Received 25 June 2001; accepted in final form 27 February 2002.
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