(Circulation. 2001;103:2967.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Role for p27Kip1 in Vascular Smooth Muscle Cell Migration
From the Cardiology Division (J.S., S.O.M, A.R.M., L.E.R.), Center for Molecular Cardiology (S.O.M., A.R.M.), Department of Medicine (H.-J.C.), and Department of Pharmacology (A.R.M.), Columbia University College of Physicians and Surgeons, and the Cardiovascular Institute, Department of Medicine, Mount Sinai School of Medicine (M.P.), New York, NY.
Correspondence to LeRoy E. Rabbani, MD, Columbia University College of Physicians and Surgeons, Division of Cardiology, 630 W 168th St, New York, NY 10032. E-mail ler8{at}columbia.edu
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Abstract |
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Background—Rapamycin is a potent inhibitor of smooth muscle cell (SMC) proliferation and migration. Rapamycin-mediated inhibition of SMC proliferation is associated with upregulation of the cyclin-dependent kinase inhibitor p27Kip1. Previously, we showed that mixed embryonic fibroblasts obtained from p27Kip1(-/-) mice were relatively rapamycin-resistant, suggesting that p27Kip1 plays an integral role in modulating the antiproliferative effects of rapamycin. We hypothesized that the antimigratory effect of rapamycin may also be mediated by p27Kip1.
Methods and Results—Rapamycin (1 to 10 nmol/L) inhibited basic fibroblast growth factor–induced migration of wild-type (WT) but not p27Kip1(-/-) SMCs in a dose-dependent manner (P<0.05) in a modified Boyden chamber. The effects of rapamycin on aortic SMC explant migration were also studied with WT, p27(+/-), and p27(-/-) mice. Rapamycin 4 mg · kg-1 · d-1 IP for 5 days inhibited SMC migration by 90% in the WT and p27Kip1(+/-) (P<0.05) but not p27Kip1(-/-) animals.
Conclusions—Lack of p27Kip1 reduces rapamycin-mediated inhibition of SMC migration. These novel findings suggest a role for p27Kip1 in the signaling pathway(s) that regulates SMC migration.
Key Words: muscle, smooth • cells • inhibitors • rapamycin
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Vascular smooth muscle cell (SMC) migration is believed to play a major role in the pathogenesis of many vascular diseases, including atherosclerosis and restenosis after PTCA and coronary stenting.1 Previously, we and others demonstrated that rapamycin, a macrolide antibiotic, inhibited SMC proliferation in vitro and in vivo by blocking cell-cycle progression at the G1/S transition.2 3 4 The inhibition of cellular proliferation was associated with a marked reduction in cyclin-dependent kinase (CDK) activity and in retinoblastoma protein phosphorylation.2 4 Pretreatment of rat and human SMCs with rapamycin (2 nmol/L) for 48 hours inhibited platelet-derived growth factor (PDGF)-induced migration in a modified Boyden chamber. Acute rapamycin treatment (6 hours) of rat and human SMCs, however, had no effect on migration, suggesting that longer exposure to rapamycin is essential for its antimigratory actions.5 The finding that rapamycin possessed both antiproliferative and antimigratory properties led us to propose that rapamycin may have important applications in the treatment of such disorders as accelerated arteriopathy that occurs in transplanted hearts and restenosis after PTCA and placement of coronary stents.2 5 6 Recently, implantation of rapamycin (sirolimus)-coated stents in de novo lesions was shown to be safe and effective in inhibiting neointimal formation at 4 months of follow-up.7
Downregulation of the CDK inhibitor (CDKI) p27Kip1 by mitogens is blocked by rapamycin.8 In p27Kip1(-/-) mice, relative rapamycin resistance was demonstrated in mixed embryonic fibroblasts and splenic T lymphocytes.9 In rapamycin-resistant myogenic cells, constitutively low levels of p27Kip1 were observed, which did not increase with serum withdrawal and rapamycin.9 Rapamycin (1 nmol/L) inhibited p70S6k phosphorylation and activity in both p27Kip1(-/-) mixed embryonic fibroblasts and rapamycin-resistant myogenic cells.9 Although rapamycin blocks p70s6k in both p27Kip1(-/-) cells and rapamycin-resistant myogenic cells, constitutively low levels or absence of p27Kip1 inhibits the antiproliferative properties of rapamycin. Decreased levels of p27Kip1 in the vessel wall have been associated with increased neointimal response after PTCA.10 Overexpression of p27Kip1 in SMCs inactivated cdk2 and cdk4 activity; adenoviral gene transfer of p27Kip1 after femoral artery balloon angioplasty significantly inhibited intimal cell proliferation.11 Similar inhibition of neointima formation has also been reported for adenovirus-mediated overexpression of p21cip1 in the rat carotid artery after angioplasty.12 In this study, we demonstrate that p27Kip1 plays a critical role in the mediation of the antimigratory properties of rapamycin.
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Methods |
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Reagents
DMEM and trypsin were from Gibco, recombinant basic fibroblast growth factor (bFGF) was from Biosource International, paclitaxel was from Sigma, and the mouse monoclonal p27Kip1 antibody was from Santa Cruz. Rapamycin was a gift from Dr Suren Sehgal (Wyeth-Ayerst Laboratories).
Expression of C3 Exoenzyme
The expression of GST-C3 exoenzyme (cDNA was a gift
from Dr Judy Meinkoth, University of Pennsylvania) was prepared as
previously
described.13 14
Cell Culture
The aortic SMCs (
5 passages) were obtained from the
explant migration experiments described below and were subcultured in
DMEM containing 20% FBS at 37°C in a humidified 95% air–5%
CO2 atmosphere. Identification of SMCs was
performed with 
-actin
immunofluorescence.
Fluorescence-Activated Cell
Sorter Analysis
Wild-type (WT)
[p27Kip1(+/+)], heterozygous
[p27Kip1(+/-)], and homozygous
[p27Kip1(-/-)] SMCs were placed in
DMEM+0.5% FBS and various concentrations of rapamycin for 48 hours to
achieve quiescence. The cells were washed, harvested, and labeled with
propidium iodide solution–RNase for 1 hour. The cells were
analyzed on a fluorescence-activated cell
sorter (FACS), with a minimum of 15 000 cells counted as previously
described.6
Assessment of Cell Viability
Trypan blue exclusion assays were performed at the
conclusion of all experiments and demonstrated >95% viability. The
effects of rapamycin (0.1 to 100 nmol/L, 48-hour incubation) on SMC
viability were also determined by a cytotoxicity assay
(Promega CytoTox 96).
SMC Adhesion Assay
SMCs were treated with rapamycin or vehicle for 48
hours. SMCs (5x105/mL in DMEM with 0.2%
BSA) were loaded onto plates precoated with laminin or fibronectin.
After 3 hours, the media containing nonadherent cells were removed, and
cell numbers were determined in triplicate with a Coulter
Counter.
SMC Migration Assay
Migration was measured by use of a 48-well modified
Boyden chamber housing a polycarbonate filter with 8-µm pores as
described.5 Rapamycin or
FK506 was added to the growth medium for 48 hours, or C3 exoenzyme was
added for 16 hours. The lower chambers were filled with either 50 ng/mL
of bFGF or 0.2% BSA (negative control) in DMEM. An equal number of
cells (2x105/mL) in 50 µL were loaded
into the top chamber of each well. After 6 hours, nonmigrating cells
were scraped from the upper surface of the filter. Cells on the lower
surface were fixed with methanol and stained with Giemsa stain. The
number of SMCs on the lower surface of the filter was determined by
counting 4 high-power (x200) fields of constant area per well. Values
are expressed as the percentage of cells migrating in response to bFGF
after subtraction of the negative control (DMEM+BSA). Experiments were
performed at least twice in quadruplicate wells.
Aortic SMC Explant Migration
The p27Kip1(+/-) and
p27Kip1(-/-) knockout mice were kindly
provided by Dr Andrew
Koff.15 WT controls were
obtained from 2 sources: C57BL/6 mice and
p27Kip1(+/+) littermates were purchased from
Jackson Laboratory (Bar Harbor, Me). No
differences in sensitivity to rapamycin were detected between the 2
groups. All mice used in these experiments were genotyped by
Southern hybridization as previously
described15 and
immunoblot analysis of
p27Kip1. The mice received 1 of 3 treatment
protocols of rapamycin: 9 mg · kg-1 ·
d-1 IP for 7 days, 4 mg ·
kg-1 · d-1
IP for 5 days, or 2 mg · kg-1 ·
d-1 IP for 2 days. The control group was
treated with vehicle alone (0.2% sodium carboxymethyl
cellulose, polysorbate 0.25%; Sigma). At the conclusion of the
protocol, the mice were euthanized with 100 mg/kg of pentobarbital, the
aortas were excised, and the adventitia and surrounding connective
tissue were removed. The aortas were opened by a longitudinal cut, and
the intima and a thin portion of the subjacent media were removed. The
descending aorta was weighed and trimmed to normalize the weight to 10
mg. The aortas were cut into 4 pieces (
2x2 mm), and each piece
was placed into a separate well of a 6-well plate containing DMEM+20%
FBS. The number of cells that explanted from each piece was counted
daily and the total number of cells/animal added. The results in Figure 4
are presented as the mean percentage (±SD) of inhibition of
migration compared with control for 
4 animals from each
group.
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Immunoblots
Lysates from SMCs growing in log phase or
treated with rapamycin (100 nmol/L for 48 hours) were prepared as
previously
described.9
Statistics
Data are presented as the mean±SD of the
independent experiments. Statistical significance was determined by
1-way ANOVA and Fisher’s protected least significant difference test.
A paired t test
(StatView 4.01) was used to analyze all
data. A value of P<0.05 was
considered statistically
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The inhibitory effects of rapamycin on the migration of SMCs isolated from WT and p27Kip1(-/-) mice were determined. In WT SMCs, rapamycin treatment for 48 hours demonstrated a significant inhibitory effect on bFGF-induced migration (Figure 1A
, open bars). The inhibition was concentration
dependent between 1 and 100 nmol/L, with an IC50
of 2 nmol/L. In contrast, no significant inhibition of migration by
rapamycin (1 to 10 nmol/L) was observed in
p27Kip1(-/-) SMCs
(Figure 1B, open bars). At higher concentrations (100
nmol/L), an 35% inhibition was observed; the
IC50 in
p27Kip1(-/-) cells was 200 nmol/L,
representing an 100-fold increased
IC50 compared with WT SMCs. Addition of
rapamycin to either the upper or lower chamber immediately before
incubation had no effect on SMC migration (data not shown). FK506, an
agent that binds to the same cytosolic receptor (FKBP12) as rapamycin,
had no effect on SMC migration
(Figure 1A and 1B, solid bars). The antimigratory effect of
rapamycin was competitively inhibited by a molar excess of FK506
(Figure 1C and 1D). These data indicate that rapamycin
inhibits SMC migration by binding to FKBP12.
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Treatment of WT SMCs with rapamycin (100 nmol/L for 48
hours) caused an increase in p27Kip1 protein
levels
(Figure 1A
, inset); in contrast, no
p27Kip1 was detected in
p27Kip1(-/-) SMCs
(Figure 1B, inset). Although rapamycin inhibits SMC
proliferation,2 these
findings cannot be explained by differences in proliferation, because
equal numbers of cells were loaded into the Boyden chamber, and the
total number of cells in the upper and lower chambers after the 6-hour
incubation was equal in the untreated and treated WT and
p27Kip1(-/-) SMCs. This is to be
expected, because the cell cycle requires 24 hours in the murine
SMCs and rapamycin inhibits cells in late G1. In
addition, we counted the number of cells in the upper chamber after 6
hours of incubation without bFGF in the lower chamber. Rapamycin had no
significant effect on the total number of cells in the upper chamber
[WT and p27Kip1(-/-)]
(P=NS)
(Figure 1E).
No differences in cell viability were noted between
untreated and rapamycin-treated SMCs obtained from WT and
p27Kip1(-/-) animals as measured by
trypan blue exclusion. To further assess cellular viability, we
determined that rapamycin (0.1 to 100 nmol/L) did not increase LDH (a
marker for cell viability) release in either WT,
p27Kip1(+/-), or
p27Kip1(-/-) SMCs
(Table).
These results indicate that the antimigratory properties of rapamycin
are not due to vascular SMC cytotoxicity. No morphological differences
were observed between untreated and rapamycin-treated (100 nmol/L for
48 hours) SMCs isolated from WT mice and
p27Kip1(-/-) mice (data not
shown).
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Because the differences in migration may be dependent on
rapamycin-induced alterations in the distribution of cells in the cell
cycle, we synchronized the vascular SMCs by serum starvation for 48
hours before loading into the modified Boyden chamber. FACS
analysis of the 3 cell types
(Figure 2A) indicated that varying concentrations of
rapamycin in addition to serum starvation did not alter the
distribution of cells in G0, S, and
G2/M phases. Under these conditions, rapamycin
treatment for 48 hours demonstrated a significantly inhibited
bFGF-induced migration in p27Kip1(+/+) and
p27Kip1(+/-) cells
(Figure 2B). The inhibition of migration was concentration
dependent between 1 and 100 nmol/L of rapamycin, with an
IC50 of 2 nmol/L. In contrast, the
IC50 for rapamycin in
p27Kip1(-/-) cells was 200 nmol/L,
representing an 100-fold increased
IC50 compared with WT SMCs
(Figure 2B). Synchronization of SMCs in
G1 by serum starvation
(Figure 2A) did not alter the antimigratory properties of
rapamycin
(Figure 2B).
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Because migration is dependent on the adhesion of the SMCs
to the Boyden chamber membrane, we performed adhesion assays with
fibronectin- and laminin-coated plates. SMCs obtained from
p27Kip1(-/-) animals demonstrated no
differences in adhesion compared with SMCs obtained from WT animals on
both fibronectin- and laminin-coated plates. Furthermore, rapamycin
treatment (100 nmol/L for 48 hours) did not affect cell adhesion in
either WT or p27Kip1(-/-) SMCs
(Figure 3).
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To assess the in vivo effects of rapamycin on SMC migration
in the p27Kip1(-/-) animals, we examined
the ability of SMCs to migrate out from aortic explants and establish
cell cultures. Rapamycin was not added to the culture medium after the
aortas were explanted. Explant migration of aortic SMCs was performed
with p27Kip1(+/+) littermates,
p27Kip1(+/-), or
p27Kip1(-/-) mice. SMCs from
p27Kip1(+/+),
p27Kip1(+/-), and
p27Kip1(-/-) mice migrated out of the
aortic explant by day 2. In animals treated with rapamycin 4 mg ·
kg-1 · d-1
IP for 5 days, 85% inhibition of migration compared with untreated
animals was observed in the p27Kip1(+/+) and
p27Kip1(+/-) groups
(P<0.05). In contrast, no
rapamycin-mediated inhibition of migration was observed in the
p27Kip1(-/-) group
(P<0.05,
Figure 4A), indicating that
p27Kip1 plays a critical role in the
rapamycin-mediated inhibition of SMC migration. At higher doses,
equivalent levels of rapamycin-mediated inhibition of migration were
observed in WT, p27Kip1(+/-), and
p27Kip1(-/-) cells
(Figure 4B). At lower doses (2 mg ·
kg-1 · d-1
for 2 days), no rapamycin-mediated inhibition of migration was observed
(data not shown). This assay, while primarily assessing SMC migration,
may also reflect proliferation, especially after several days of
culture. These results are consistent with the findings
obtained in the modified Boyden chamber for
p27Kip1(-/-) cells and suggest that both
p27Kip1-dependent and
p27Kip1-independent pathways mediate the SMC
antimigratory actions of rapamycin. To demonstrate that
p27Kip1(-/-) SMC migration could be
inhibited by agents that act independently of the
p27Kip1 pathway, we treated WT and
p27Kip1(-/-) mice with taxol 20 mg ·
kg-1 · d-1
IP for 7 days.16 No
differences in taxol-mediated inhibition were observed in the 2 groups
(Figure 4C).
Recent data suggest that the Ras/RhoA mitogenic
pathway regulates the degradation of
p27Kip1.17
C3 exoenzyme, which ADP-ribosylates and inactivates RhoA,
inhibited PDGF-induced p27Kip1 degradation.
These findings suggest that activation of RhoA by mitogens is necessary
for degradation of
p27Kip1.17
In addition, thrombin-induced vascular SMC DNA synthesis and migration
were inhibited by C3
exoenzyme.14 We sought to
determine whether this inhibition of migration was mediated, in part,
by regulating p27Kip1 levels. SMCs from WT
and p27Kip1(-/-) animals were exposed to
either 2 or 20 µg/mL for 16 hours, trypsinized, and loaded into the
upper chamber of the Boyden chamber. Higher concentrations of C3
exoenzyme (40 µg/mL) were toxic to the cells, because we observed
increased trypan blue uptake; therefore, higher concentrations of C3
exoenzyme were not used in these experiments. C3 exoenzyme
significantly inhibited bFGF-mediated SMC migration in WT cells
(Figure 5, open bars). SMCs from
p27Kip1(-/-) animals demonstrated a 25%
relative resistance to C3 exoenzyme
(Figure 5, solid bars). SMCs that were acutely exposed to C3
exoenzyme demonstrated no inhibition of migration. These results
implicate p27Kip1 as a regulator, in part,
of both rapamycin- and C3 exoenzyme–mediated inhibition of SMC
migration.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Rapamycin has been shown to inhibit rat, porcine, and human SMC migration.5 In addition, rapamycin reduces intimal thickening by 50% after PTCA in a porcine model.4 The rapamycin antirestenotic effect is characterized by an inhibition of the SMC response to coronary injury with a concomitant decrease in retinoblastoma protein phosphorylation as well as an increase in p27Kip1 levels, thereby resulting in cell-cycle arrest.2 4
The data indicate that the CDKI
p27Kip1 plays a critical role in mediating
the antiproliferative9 and
antimigratory properties of rapamycin. The lack of
p27Kip1 in the null mice was associated with
an 100-fold increase in the IC50 for
migration compared with WT SMCs in Boyden chamber assays
(Figures 1A, 1B, and 2B). Similarly, when aortic explants of
mice treated with rapamycin 4 mg · kg-1
· d-1 for 5 days were used,
p27Kip1(-/-) mice demonstrated
significantly less inhibition of SMC migration than WT animals. At
higher concentrations of rapamycin (9 mg ·
kg-1 · d-1
IP for 7 days), no significant difference was observed between
p27Kip1(-/-) and WT animals. These
findings suggest that at higher doses and duration of treatment,
p27Kip1-independent actions of rapamycin can
inhibit migration. Both p27Kip1(-/-) and
WT animals demonstrated no inhibition of migration after lower doses of
rapamycin (2 mg · kg-1 ·
d-1 for 2 days). This is
consistent with previous reports in rats demonstrating that low
doses of rapamycin (1.5 mg · kg-1 for
14 days) had no effect on alloimmune injury and only a 45% reduction
in intimal hyperplasia after mechanical injury. In contrast, higher
concentrations of rapamycin (6 mg · kg-1
· d-1 for 7 days followed by 3
mg · kg-1 for 7 days) diminished
intimal thickening by
98%.18 Mice treated with
rapamycin 6 mg · kg-1 ·
d-1 for 14 days showed no evidence of
renal, cardiac, or liver damage, and marrow cellularity was
normal.19
We have previously shown that splenic T lymphocytes derived
from p27Kip1(-/-) mice required a 15- to
30-fold higher concentration of rapamycin than do WT cells for a
comparable level of growth
inhibition.9 The inhibition
of migration in both WT and p27Kip1(-/-)
SMCs was inhibited by excess FK506, implying that the antimigratory
properties of rapamycin are mediated by FKBP12. Similarly, we have
previously demonstrated that the antiproliferative properties of
rapamycin are mediated through FKBP12
binding.2 Approximately 20%
of WT SMCs were resistant to the antimigratory effects of
rapamycin; this is consistent with findings that suggested that
10% to 20% of rat aortic SMCs were resistant to the
antiproliferative effects of
rapamycin.2 In BC3H1 myogenic
cells, we were able to enrich for this population by serially passaging
these cells in the presence of rapamycin (100 or 1000 nmol/L).
Interestingly, these cells demonstrated constitutively low levels of
p27Kip1, which did not increase on serum
withdrawal and treatment with
rapamycin.9 SMCs derived from
p27Kip1(-/-) animals did not demonstrate
increased proliferation9 or
migration in Boyden chamber or explant assays.
Rapamycin (100 nmol/L) inhibited WT SMC migration by
80%, compared with 35% in
p27Kip1(-/-) cells. Thus, in a subset of
SMCs (35%), rapamycin inhibits migration via a pathway that is
independent of p27Kip1. Indeed, rapamycin
has multiple effects on cell signaling
(Figure 6). A common step is binding of the FK506 binding
protein (FKBP12) to the cytosolic receptor of
rapamycin.20
Rapamycin-FKBP12 also inhibits the mammalian target of rapamycin
(mTOR),21
p70S6k,22
and 4E-BP1
phosphorylation.23
FK506 competes for binding to FKBP12 with rapamycin, but FK506 does not
inhibit mTOR, p70S6k, or 4E-4BP1. Molar
excesses of FK506 compete rapamycin off from FKBP12 and block the
inhibition of migration by rapamycin in
p27Kip1(-/-) SMCs, indicating that both
the p27Kip1-dependent and
p27Kip1-independent antimigratory properties
of rapamycin are mediated through FKBP12
(Figure 6). p70s6k
phosphorylation and activity, however, are completely
inhibited by 1 nmol/L rapamycin in WT and
p27Kip1(-/-)
cells.9 These findings
suggest that in a subset of SMCs, rapamycin inhibits migration via a
p70s6k- and
p27Kip1-independent pathway that has yet to
be identified
(Figure 6).
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p27Kip1 levels are regulated in
part by the Ras/RhoA mitogenic pathway
(Figure 6). Overexpression of a dominant negative Ras or RhoA
inhibits PDGF-induced degradation of
p27Kip1. C3 exoenzyme, which ADP-ribosylates
and inactivates RhoA, inhibits PDGF-induced
p27Kip1
degradation17 24
and inhibits thrombin-mediated vascular SMC proliferation and
migration.14 Because loss of
p27Kip1 reduced C3 exoenzyme (20 µg/mL)
inhibition by 25%, the Rho pathway mediates migration through both
p27Kip1-dependent and independent pathways.
Higher concentrations of C3 exoenzyme led to decreased cell viability.
In Swiss 3T3 fibroblasts, it has been shown that Rho can be
activated by extracellular ligands and that Rho activation can
lead to the assembly of contractile actin-myosin filaments and focal
adhesion complexes.25 These
observations suggest that the Rho GTPase family is one of the key
regulatory molecules that link surface receptors to the organization of
the actin cytoskeleton. Rapamycin has not been shown to interact with
the Rho GTPase family, although it is interesting that inhibition of
Rho17 24 and of
mTOR8 26 are both
associated with increased levels of the CDKI
p27Kip1
(Figure 6).
The function of p27Kip1 is clinically relevant because of the connections that have been made between the downregulation and enhanced degradation of p27Kip1 in colorectal, stomach, breast, and small-cell lung cancers.27 Our findings suggest that agents that increase p27Kip1 levels in vivo may have both an antiproliferative and antimigratory effect. Rapamycin-mediated inhibition of SMC migration appears to depend on the presence of p27Kip1. This intriguing finding implicates p27Kip1 in the signaling pathway(s) that regulates both SMC proliferation and migration. Pharmacological and/or recombinant technologies aimed at increasing p27Kip1 may have dramatic effects on the amelioration of restenosis after angioplasty/stent placement and accelerated arteriopathy after cardiac transplantation.
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Acknowledgments |
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This work was supported by the Sol and Margaret Berger Foundation, Clifton, NJ (Dr Rabbani); an American Heart Association Clinician-Scientist Award to Dr Marx; and NIH grants R01-HL-56180, R01-HL-30290, R01-A1-39794, and R03-TW-00949 and a Johnson and Johnson focused giving grant to Dr Marks. Dr Marks is the Clyde and Helen Wu Professor of Molecular Cardiology and a Doris Duke Distinguished Clinical Scientist. We gratefully acknowledge Dr Andrew Koff for generously providing the p27-null mice used in this study.
Received December 8, 2000; revision received February 7, 2001; accepted February 19, 2001.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Schwartz SM. Smooth muscle migration in atherosclerosis and restenosis. J Clin Invest. 1997;100:S87–S98.
2.
Marx SO, Jayaraman
T, Go LO, et al. Rapamycin-FKBP inhibits cell cycle regulators of
proliferation in vascular smooth muscle cells.
Circ Res. 1995;76:412–417.
3.
Poston RS,
Billingham M, Hoyt EG, et al. Rapamycin reverses chronic graft vascular
disease in a novel cardiac allograft model.
Circulation. 1999;100:67–74.
4.
Gallo R, Padurean
A, Jayaraman T, et al. Inhibition of intimal thickening after balloon
angioplasty in porcine coronary arteries by targeting
regulators of the cell cycle.
Circulation. 1999;99:2164–2170.
5. Poon M, Marx SO, Gallo R, et al. Rapamycin inhibits vascular smooth muscle cell migration. J Clin Invest. 1996;98:2277–2283.[Medline] [Order article via Infotrieve]
6.
Marx SO, Marks AR.
Cell cycle progression and proliferation despite 4BP-1
dephosphorylation. Mol
Cell Biol. 1999;19:6041–6047.
7.
Sousa JE, Costa MA,
Abizaid A, et al. Lack of neointimal proliferation after
implantation of sirolimus-coated stents in human coronary
arteries. Circulation. 2001;103:192–195.
8. Nourse J, Firpo E, Flanagan WM, et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994;372:570–573.[Medline] [Order article via Infotrieve]
9.
Luo Y, Marx SO,
Kiyokawa H, et al. Rapamycin resistance tied to defective regulation of
p27Kip1. Mol
Cell Biol. 1996;16:6744–6751.
10.
Tanner FC, Yang
ZY, Duckers E, et al. Expression of cyclin-dependent kinase
inhibitors in vascular disease.
Circ Res. 1998;82:396–403.
11.
Tanner FC, Boehm
M, Akyurek LM, et al. Differential effects of the cyclin-dependent
kinase inhibitors p27Kip1,
p21cip1, and
p16ink4 on vascular smooth muscle cell
proliferation. Circulation. 2000;101:2022–2025.
12. Chang MW, Barr E, Lu MM, et al. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96:2260–2268.
13. Dillon ST, Feig LA. Purification and assay of recombinant C3 transferase. Methods Enzymol. 1995;256:174–184.[Medline] [Order article via Infotrieve]
14.
Seasholtz TM,
Majumdar M, Kaplan DD, et al. Rho and Rho kinase mediate
thrombin-stimulated vascular smooth muscle cell DNA synthesis and
migration. Circ Res. 1999;84:1186–1193.
15. Kiyokawa H, Kineman RD, Manova-Todorova KO, et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell. 1996;85:721–732.[Medline] [Order article via Infotrieve]
16. Sollott SJ, Cheng L, Pauly RR, et al. Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat. J Clin Invest. 1995;95:1869–1876.
17.
Weber JD, Hu W,
Jefcoat SC, et al. Ras-stimulated extracellular signal-related kinase1
and RhoA activities coordinate platelet-derived growth
factor-induced G1 progression through the independent regulation of
cyclin D1 and p27Kip1.
J Biol Chem. 1997;272:32966–32971.
18. Gregory C, Huie P, Billingham M, et al. Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Transplantation. 1993;55:1409–1418.[Medline] [Order article via Infotrieve]
19. Morris RE. Rapamycins: antifungal, antitumor, antiproliferative, and immunosuppressive macrolides. Transplant Rev. 1992;6:39–87.
20.
Schreiber S.
Chemistry and biology of the immunophilins and their immunosuppressive
ligands. Science. 1991;251:283–287.
21. Brown E, Albers T, Shin T, et al. A mammalian protein targeted by G1-arresting rapamycin complex. Nature. 1994;369:756–758.[Medline] [Order article via Infotrieve]
22. Brown EJ, Beal PA, Keith CT, et al. Control of p70 S6 kinase by kinase activity of FRAP in vivo. Nature. 1995;377:441–446.[Medline] [Order article via Infotrieve]
23.
Brunn GJ, Hudson
CC, Sekulic A, et al. Phosphorylation of the
translational repressor PHAS-I by the mammalian target of rapamycin.
Science. 1997;277:99–101.
24.
Hirai A, Nakamura
S, Noguchi Y, et al. Geranylgeranylated rho small GTPase(s) are
essential for the degradation of p27 Kip1 and facilitate the
progression from G1 to S phase in growth-stimulated rat FRTL-5 cells.
J Biol Chem. 1997;272:13–16.
25.
Hall A. Rho
GTPases and the actin cytoskeleton.
Science. 1998;279:509–514.
26. Sabatini DM, Erdjument-Bromage H, Lui M, et al. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell. 1994;78:35–43.[Medline] [Order article via Infotrieve]
27. Steeg PS, Abrams JS. Cancer prognostics: past, present and p27. Nat Med. 1997;3:152–154. [Medline] [Order article via Infotrieve]
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|
|
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|
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|
|
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|
|
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|
|
|
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