Bradykinin B1 receptor blocks PDGF-induced mitogenesis by prolonging ERK activation and increasing p27Kip1

Bradley S. Dixon, David Evanoff, Wei B. Fang, and Michael J. Dennis

Division of Nephrology, Department of Medicine, Department of Veterans Affairs Medical Center and University of Iowa College of Medicine, Iowa City, Iowa 52242-1081


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals. DABK was obtained from Bachem (Torrance, CA); recombinant human PDGF-BB, recombinant human interleukin (IL)-1beta , 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 [gamma -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-1beta (25). In the absence of cytokines, expression of the B1R was found to be variable and occasionally absent (unpublished data). Therefore, unless otherwise stated, the cells were pretreated with IL-1beta (2 ng/ml) in serum-free medium for 4-24 h to upregulate expression of B1R before addition of the experimental agents. Our previous studies (11) showed that DABK could be added up to 8 h after PDGF to produce complete inhibition of mitogenesis. Therefore, in these studies, unless otherwise stated DABK was added 1 h after addition of PDGF. For experiments that used inhibitors of MEK or Src family kinase (SFK), the inhibitors were added 30 min after treatment with PDGF but 30 min before addition of DABK. This allowed us to focus the effect of the inhibitor on DABK-stimulated events without inhibiting early PDGF-stimulated events.

Immunoprecipitation 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 beta -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 beta -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 [gamma -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-1beta (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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   des-Arg9-bradykinin (DABK) inhibits platelet-derived growth factor (PDGF)-stimulated activation of Cdk2. Confluent, serum-deprived vascular smooth muscle cells were pretreated with vehicle (0) or interleukin (IL)-1beta for 4 h before addition of either PDGF (15 ng/ml) or PDGF (15 ng/ml) + DABK (100 nM) for the times noted. The cells were then extracted, and immunoprecipitation (IP) kinase assays were performed as described in EXPERIMENTAL PROCEDURES after immunoprecipitation with an antibody against Cdk2, cyclin E, or cyclin A as noted.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of DABK on G1-phase cell cycle proteins. Confluent, serum-deprived vascular smooth muscle cells were pretreated with vehicle (0) or IL-1beta for 4 h before addition of either PDGF (15 ng/ml) or PDGF (15 ng/ml) + DABK (100 nM) for the times noted. The cells were then extracted and resolved by SDS-PAGE and immunoblot assays performed as described in EXPERIMENTAL PROCEDURES for each of the G1-phase cell cycle proteins noted. A: a representative immunoblot for each of the proteins. pRb and Rb denote the hyperphosphorylated and hypophosphorylated forms of retinoblastoma protein (Rb), respectively (13). The blot for Cdk2 (bottom) was loaded on the gel in a slightly different pattern than the preceding blots. It demonstrates that stimulation with PDGF produced a more rapid mobility band characteristic of the phosphorylated active form of Cdk2 and this was blocked by treatment with DABK (30). B: quantitative results from 6 independent experiments. The results are expressed as % maximal value in each experiment, and the results at each time point are combined to obtain means ± SE (* P < 0.05 for differences between PDGF + DABK compared with PDGF alone).

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.


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3.   DABK increases the association of p27Kip1 with cyclin E-Cdk2. Confluent, serum-deprived vascular smooth muscle cells were pretreated with IL-1beta for 12 h before treatment with vehicle (basal, B), 15 ng/ml PDGF (P), or 15 ng/ml PDGF + 100 nM DABK (P+D) for 12 h. Cells were extracted, and total p27Kip1 was measured in the lysate by immunoblotting (top). The remaining lysate was immunoprecipitated with an antibody against cyclin E and used either to measure Cdk2 activity (middle) as described in EXPERIMENTAL PROCEDURES or immunoblotted (bottom) for p27Kip1 and cyclin E in the immunoprecipitated pellet (Pt) or remained in the supernatant after immunoprecipitation (Sup). The experiments were repeated 3 times with the same results.

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.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 4.   DABK prolongs PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase kinase (MEK). Confluent, serum-deprived vascular smooth muscle cells were pretreated with IL-1beta for 12 h before treatment with vehicle (0) or PDGF (15 ng/ml), DABK (100 nM), or PDGF + DABK for the times indicated. After treatment, cells were extracted into lysis buffer and the active phosphorylated forms of p44/p42 ERK (phospho-p44 or -p42) as well as total p42 ERK were measured with specific antibodies as described in EXPERIMENTAL PROCEDURES. A represents short-term stimulation and demonstrates that treatment with PDGF + DABK prolonged activation of ERK at 2 and 4 h compared with either PDGF or DABK alone. The slower mobility band in the blots stained for total ERK represents the phosphorylated active ERK and reproduces the pattern seen in blots with the phosphospecific antibody. The results were confirmed in 3 separate experiments. B and C demonstrate the effect of treatment with PDGF and PDGF + DABK on the active phosphorylated and total form of ERK (B) and MEK (C) at longer time points corresponding to treatments shown in Figs. 1 and 2. The results for phospho-p44 ERK (pp44) and phospho-MEK (pMEK) from 4 separate experiments were quantitated by densitometry, and the means ± SE are plotted (* P < 0.01 compared with PDGF alone). Similar results were obtained for phospho-p42 ERK.

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.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   MEK and Src family kinase (SFK) inhibitors block DABK-mediated activation of ERK. Confluent, serum-deprived vascular smooth muscle cells were pretreated with IL-1beta for 12 h. Cells were then treated with either serum-free medium (basal, B) or 15 ng/ml PDGF (P) for 30 min before addition of vehicle (None), one of the MEK inhibitors (50 µM PD-98059 or 5 µM U-0126), or the SFK inhibitor (1 µM PP2) as noted. After an additional 30 min, vehicle or 100 nM DABK (D) was added and the incubation continued for a total of 4 h beyond the addition of PDGF. The cells were extracted at 4 h after addition of PDGF or vehicle for measurement of the active phosphorylated forms of p44/p42 ERK and total p42 ERK with specific antibodies as described in EXPERIMENTAL PROCEDURES. The results are representative of 2 experiments.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition of either MEK or a SFK prevents upregulation of p27Kip1 but not downregulation of cyclin D by DABK. Confluent, serum-deprived vascular smooth muscle cells were pretreated with IL-1beta for 12 h. Cells were then treated as described in Fig. 5 except that the incubation was continued for a total of 12 h after addition of PDGF. The cells were then extracted, and expression of p27Kip1 (A), cyclin D1 (B), cyclin D2 (C), or cyclin D3 (D) was detected by immunoblotting and quantitated by densitometry as described in EXPERIMENTAL PROCEDURES. The results are expressed as %control, where the control was the level of expression seen in the vehicle-only treated cells (Basal) not exposed to any inhibitor (None) shown by the first open bar in each graph. Each data point represents the mean ± SE from at least 6 independent experiments (* P < 0.05 for effect of PDGF+DABK compared with PDGF for each inhibitor). The MEK inhibitors U-0126 and PD-98059, but not the SFK inhibitor PP2, significantly increased the expression of p27Kip1, cyclin D1, and cyclin D2 in quiescent (Basal) cells compared with cells that did not receive the inhibitor (P < 0.05).

We also examined the effect of the MEK inhibitors on the expression of cyclin D. The same experimental paradigm was employed as for p27Kip1, and the results are shown in Fig. 6, B-D. Inhibition of MEK led to increased basal levels of cyclin D1 and D2 in quiescent cells and partially inhibited the upregulation of all isoforms of cyclin D by PDGF. However, in contrast to the effect on p27Kip1, inhibition of MEK did not prevent DABK from inhibiting the expression of either cyclin D2 or D3 [percent inhibition of PDGF-stimulated cyclin D2 expression by DABK after treatment with vehicle (52.5 ± 7.1%), PD-98059 (62.1 ± 22.7%), or U-0126 (102.6 ± 22.3%), P = not significant (NS); percent inhibition of PDGF-stimulated cyclin D3 by DABK after treatment with vehicle (71.9 ± 10%), PD-98059 (93.8 ± 34.2%), or U-0126 (64.1 ± 7.5%), P = NS]. These studies again demonstrate that inhibition of MEK has different effects in quiescent and mitogen-stimulated cells and confirm that inhibition of MEK had the expected effect to inhibit PDGF-stimulated expression of cyclin D. The results further imply that DABK uses a pathway other than activation of ERK to inhibit the PDGF-stimulated expression of cyclin D.

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.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   MEK inhibitors partially reverse the antiproliferative effects of DABK. Confluent, serum-deprived vascular smooth muscle cells were pretreated with IL-1beta for 12 h and then treated exactly as described in Fig. 6. Vehicle (None), the MEK inhibitors PD-98059 (50 µM) or U-0126 (5 µM), or the SFK inhibitor PP2 (1 µM) was added 30 min after PDGF (15 ng/ml) and 30 min before addition of DABK (100 nM). Basal, cells that did not receive either PDGF or PDGF+DABK. [3H]thymidine (0.5 µCi/well) was added immediately after DABK, and the incubation continued for an additional 32 h before extraction of DNA for measurement of [3H]thymidine incorporation as described in EXPERIMENTAL PROCEDURES. Data are expressed as mean (± SE) cpm/well from 16 individual measurements obtained from 2 different cell preparations. Similar results were obtained from an additional 2 experiments in which the inhibition produced by DABK was 100%. Percent inhibition produced by DABK was calculated from the cpm data with the equation [(cpmPDGF - cpmPDGF+DABK)/(cpmPDGF - cpmBasal)] × 100. Treatment with PD-98059, U-0126, and PP2 partially reversed the DABK-mediated inhibition of PDGF-stimulated mitogenesis (* P < 0.001 for %inhibition by DABK after treatment with each inhibitor compared with the vehicle-treated control).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of DABK on thymidine incorporation and cell cycle proteins

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   Model for the hormonal regulation of p27Kip1 and cyclin D by ERK in arterial smooth muscle cells. Sequential activation of cyclin D-Cdk4 and cyclin E-Cdk2 promotes progression from G1 into S phase. p27Kip1 inhibits the activity of cyclin E-Cdk2 but promotes activation of cyclin D-Cdk4. Hormones such as angiotensin II (ANG II) that stimulate ERK can promote upregulation of both cyclin D and p27Kip1. The increase in p27Kip1 inhibits cyclin E-Cdk2 and entry into S phase but is permissive for the development of cell hypertrophy. Stimulation by PDGF promotes entry into S phase by activating signaling pathways in addition to ERK [e.g., phosphatidylinositol-3-kinase (PI3K)] that override the effect of ERK and inhibit p27Kip1 expression. Treatment with DABK inhibits PDGF-stimulated mitogenesis by prolonging the PDGF-mediated activation of ERK, leading to increased p27Kip1. In addition, DABK activates a separate unknown pathway (not activated by ANG II) that inhibits expression of cyclin D. This prevents cyclin D from titrating p27Kip1 away from inhibition of cyclin E-Cdk2.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Agata, J, Miao RQ, Yayama K, Chao L, and Chao J. Bradykinin B1 receptor mediates inhibition of neointima formation in rat artery after balloon angioplasty. Hypertension 36: 364-370, 2000[Abstract/Free Full Text].

2.   Agrawal, D, Hauser P, McPherson F, Dong F, Garcia A, and Pledger WJ. Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells. Mol Cell Biol 16: 4327-4336, 1996[Abstract].

3.   Aktas, H, Cai H, and Cooper GM. Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Mol Cell Biol 17: 3850-3857, 1997[Abstract].

4.   Alessi, DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489-27494, 1995[Abstract/Free Full Text].

5.   Braun-Dullaeus, RC, Mann MJ, Ziegler A, von der Leyen HE, and Dzau VJ. A novel role for the cyclin-dependent kinase inhibitor p27(Kip1) in angiotensin II-stimulated vascular smooth muscle cell hypertrophy. J Clin Invest 104: 815-823, 1999[Abstract/Free Full Text].

6.   Busse, D, Doughty RS, Ramsey TT, Russell WE, Price JO, Flanagan WM, Shawver LK, and Arteaga CL. Reversible G1 arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity. J Biol Chem 275: 6987-6995, 2000[Abstract/Free Full Text].

7.   Cheng, M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, and Sherr CJ. The p21(Cip1) and p27(Kip1) CDK "inhibitors" are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 18: 1571-1583, 1999[Abstract/Free Full Text].

8.   Cheng, M, Sexl V, Sherr CJ, and Roussel MF. Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1). Proc Natl Acad Sci USA 95: 1091-1096, 1998[Abstract/Free Full Text].

9.   Della Rocca, GJ, van Biesen T, Daaka Y, Luttrell DK, Luttrell LM, and Lefkowitz RJ. Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase. J Biol Chem 272: 19125-19132, 1997[Abstract/Free Full Text].

10.   Dixon, BS, Breckon R, Fortune J, Vavrek RJ, Stewart JM, Marzec-Calvert R, and Linas SL. Effects of kinins on cultured arterial smooth muscle. Am J Physiol Cell Physiol 258: C299-C308, 1990[Abstract/Free Full Text].

11.   Dixon, BS, and Dennis MJ. Regulation of mitogenesis by kinins in arterial smooth muscle cells. Am J Physiol Cell Physiol 273: C7-C20, 1997[Abstract/Free Full Text].

12.   Dixon, BS, Sharma RV, Dickerson T, and Fortune J. Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle. Am J Physiol Cell Physiol 266: C1406-C1420, 1994[Abstract/Free Full Text].

13.   Driscoll, B, T'Ang A, Hu YH, Yan CL, Fu Y, Luo Y, Wu KJ, Wen S, Shi XH, Barsky L, Weinberg K, Murphree AL, and Fung YK. Discovery of a regulatory motif that controls the exposure of specific upstream cyclin-dependent kinase sites that determine both conformation and growth suppressing activity of pRb. J Biol Chem 274: 9463-9471, 1999[Abstract/Free Full Text].

14.   Emanueli, C, Bonaria Salis M, Figueroa C, Chao J, Chao L, Gaspa L, Capogrossi MC, and Madeddu P. Participation of kinins in the captopril-induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse. Br J Pharmacol 130: 1076-1082, 2000[Abstract/Free Full Text].

15.   Emanueli, C, Salis MB, Chao J, Chao L, Agata J, Lin KF, Munao A, Straino S, Minasi A, Capogrossi MC, and Madeddu P. Adenovirus-mediated human tissue kallikrein gene delivery inhibits neointima formation induced by interruption of blood flow in mice. Arterioscler Thromb Vasc Biol 20: 1459-1466, 2000[Abstract/Free Full Text].

16.   Favata, MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632, 1998[Abstract/Free Full Text].

17.   Geisterfer, AA, Peach MJ, and Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 62: 749-756, 1988[Abstract].

18.   Greulich, H, and Erikson RL. An analysis of Mek1 signaling in cell proliferation and transformation. J Biol Chem 273: 13280-13288, 1998[Abstract/Free Full Text].

19.   Ishida, M, Ishida T, Thomas SM, and Berk BC. Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circ Res 82: 7-12, 1998[Abstract/Free Full Text].

20.   Kawada, M, Yamagoe S, Murakami Y, Suzuki K, Mizuno S, and Uehara Y. Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway. Oncogene 15: 629-637, 1997[ISI][Medline].

21.   Kerkhoff, E, and Rapp UR. Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1. Mol Cell Biol 17: 2576-2586, 1997[Abstract].

22.   Kerkhoff, E, and Rapp UR. Cell cycle targets of Ras/Raf signalling. Oncogene 17: 1457-1462, 1998[ISI][Medline].

23.   LaBaer, J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, and Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev 11: 847-862, 1997[Abstract].

24.   Lavoie, JN, L'Allemain G, Brunet A, Muller R, and Pouyssegur J. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 271: 20608-20616, 1996[Abstract/Free Full Text].

25.   Marceau, F, Hess JF, and Bachvarov DR. The B1 receptors for kinins. Pharmacol Rev 50: 357-386, 1998[Abstract/Free Full Text].

26.   Medema, RH, Kops GJ, Bos JL, and Burgering BM. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404: 782-787, 2000[ISI][Medline].

27.   Montagnoli, A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, and Pagano M. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev 13: 1181-1189, 1999[Abstract/Free Full Text].

28.   Morgan, DO. Principles of CDK regulation. Nature 374: 131-134, 1995[ISI][Medline].

29.   Polyak, K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, and Koff A. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev 8: 9-22, 1994[Abstract].

30.   Polyak, K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, and Massague J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78: 59-66, 1994[ISI][Medline].

31.   Pruneau, D, Luccarini JM, Robert C, and Belichard P. Induction of kinin B1 receptor-dependent vasoconstriction following balloon catheter injury to the rabbit carotid artery. Br J Pharmacol 111: 1029-1034, 1994[Abstract].

32.   Pumiglia, KM, and Decker SJ. Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 94: 448-52, 1997[Abstract/Free Full Text].

33.   Raidoo, DM, Ramsaroop R, Naidoo S, Muller-Esterl W, and Bhoola KD. Kinin receptors in human vascular tissue: their role in atheromatous disease. Immunopharmacology 36: 153-160, 1997[ISI][Medline].

34.   Rivard, N, Boucher MJ, Asselin C, and L'Allemain G. MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. Am J Physiol Cell Physiol 277: C652-C664, 1999[Abstract/Free Full Text].

35.   Schieffer, B, Drexler H, Ling BN, and Marrero MB. G protein-coupled receptors control vascular smooth muscle cell proliferation via pp60c-src and p21ras. Am J Physiol Cell Physiol 272: C2019-C2030, 1997[Abstract/Free Full Text].

36.   Servant, MJ, Coulombe P, Turgeon B, and Meloche S. Differential regulation of p27(Kip1) expression by mitogenic and hypertrophic factors: involvement of transcriptional and posttranscriptional mechanisms. J Cell Biol 148: 543-556, 2000[Abstract/Free Full Text].

37.   Sherr, CJ. Cancer cell cycles. Science 274: 1672-1677, 1996[Abstract/Free Full Text].

38.   Sherr, CJ, and Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501-1512, 1999[Free Full Text].

39.   Weber, JD, Hu W, Jefcoat SC, Jr, Raben DM, and Baldassare JJ. Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27. J Biol Chem 272: 32966-32971, 1997[Abstract/Free Full Text].

40.   Wilkie, N, Morton C, Ng LL, and Boarder MR. Stimulated mitogen-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II. A role for phospholipase D. J Biol Chem 271: 32447-32453, 1996[Abstract/Free Full Text].

41.   Winston, J, Dong F, and Pledger WJ. Differential modulation of G1 cyclins and the Cdk inhibitor p27kip1 by platelet-derived growth factor and plasma factors in density-arrested fibroblasts. J Biol Chem 271: 11253-11260, 1996[Abstract/Free Full Text].

42.   Wolf, G, and Stahl RA. Angiotensin II-stimulated hypertrophy of LLC-PK1 cells depends on the induction of the cyclin-dependent kinase inhibitor p27Kip1. Kidney Int 50: 2112-2119, 1996[ISI][Medline].

43.   Woods, D, Parry D, Cherwinski H, Bosch E, Lees E, and McMahon M. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol Cell Biol 17: 5598-5611, 1997[Abstract].

44.   Yue, J, Buard A, and Mulder KM. Blockade of TGFbeta3 up-regulation of p27Kip1 and p21Cip1 by expression of RasN17 in epithelial cells. Oncogene 17: 47-55, 1998[ISI][Medline].

45.   Zerfass-Thome, K, Schulze A, Zwerschke W, Vogt B, Helin K, Bartek J, Henglein B, and Jansen-Durr P. p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression. Mol Cell Biol 17: 407-415, 1997[Abstract].


Am J Physiol Cell Physiol 283(1):C193-C203




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
283/1/C193    most recent
00289.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Dixon, B. S.
Articles by Dennis, M. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Dixon, B. S.
Articles by Dennis, M. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online