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Departments of Pharmacology (D.K., S.A.S., R.K.A.) and Medicine (D.J.R.) and Center for Experimental Therapeutics (E.M.S.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; Wistar Institute, Philadelphia, Pennsylvania (I.A., E.P.); and The Ludwig Institute for Cancer Research, New York, New York (E.P.)
Received February 5, 2003; accepted May 13, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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), which
can inhibit platelet adhesion and aggregation as well as smooth muscle cell
proliferation (Fink et al.,
1999). Injury to the endothelium, or disturbances in laminar flow,
may reduce the levels of PGI2 by inhibiting production of
cyclooxygenase-2 (Topper et al.,
1996), the predominant source of PGI2 biosynthesis in
humans (McAdam et al., 1999).
PGI2 signals through its interaction with IP, a heterotrimeric
G-protein-coupled receptor (Coleman et al.,
1994), and others have demonstrated that the deletion of IP in
mice exacerbates injury-induced smooth muscle cell proliferation and neointima
formation (Cheng et al., 2002),
key features of restenosis and atherosclerotic plaque formation.
IP couples to adenylyl cyclase through Gs
(Namba et al., 1994) but can
also regulate other effector systems by coupling to G proteins such as
Gi and Gq (Schwaner
et al., 1995; Smyth et al.,
1996; Lawler et al.,
2001). Lawler et al.
(2001) have reported that the
mouse IP couples initially to Gs, followed by a switch in coupling
to Gi and Gq, as has also been observed for the
2-adrenergic receptor (Daaka et al.,
1997). This receptor switch has been attributed to protein kinase
A-mediated receptor phosphorylation. However, other kinases, including protein
kinase C, AKT, and casein kinase 1
, can also phosphorylate G-protein
coupled receptors (Smyth et al.,
1996; Lee et al.,
2001; Tobin,
2002). These phosphorylation events may play important roles in
receptor-mediated signaling pathways.
PGI2 inhibits the proliferation of cultured primary arterial
smooth muscle cells by blocking progression from G1 to S phase of
the cell cycle (Weber et al.,
1998). G1 phase cell cycle progression is regulated by
a sequential activation of cyclin-dependent kinases (cdks), namely cyclin
D1-cdk4/6 and cyclin E-cdk2. The activation of cyclin D1-cdk4/6 is regulated
primarily through the induction of cyclin D1, whereas the activation of cyclin
E-cdk2 is regulated primarily through a decreased association of the cdk
inhibitors, usually p21Cip1 and
p27Kip1
(Sherr and Roberts, 1999). In
large part, active cyclin D1-cdk4/6 and cyclin E-cdk2 are thought to regulate
G1 phase progression by phosphorylating pocket proteins (pRb, p107,
and p130), thereby regulating the activation of E2F-dependent genes such as
cyclin A (Takahashi et al.,
2000). Cyclin A is induced at the G1/S interface, and
the consequent formation of cyclin A-cdk2 complexes marks entry into S phase
of the cell cycle.
Several reports show that cyclin A gene expression is regulated by
E2F-pocket protein complexes. For example, overexpression of E2F-1 and human
papilloma virus E7 rescues cyclin A expression and anchorage-independent
growth (Schulze et al., 1998).
Regulation of the cyclin A promoter by pocket proteins seems to involve two
contiguous cis elements: the cell cycle-dependent element (CDE) and
the cell cycle gene homology region (CHR)
(Zwicker et al., 1995;
Liu et al., 1998). The
interactions of E2Fs and pocket proteins on the cyclin A promoter and the
mechanisms by which these proteins control cyclin A expression are topics of
active investigation (Takahashi et al.,
2000; Wu et al.,
2001), but remain poorly understood.
The cyclin A promoter contains several regulatory elements in addition to
the CDE/CHR, including the well documented cAMP response element (CRE) and
CCAAT element (Desdouets et al.,
1995; Kramer et al.,
1996). The CRE, CCAAT and CDE/CHR elements are clustered within a
118-base pair region near the transcription start sites
(Henglein et al., 1994;
Shimizu et al., 1998). Several
studies show that the CRE and CCAAT sites are necessary for the efficient
activation of the cyclin A gene (Desdouets
et al., 1995; Kramer et al.,
1996; Bottazzi et al.,
2001). The CRE-binding protein (CREB) family of transcription
factors, including CREB, ATF-1, and CRE modulator, function in a variety of
physiological processes and are activated by phosphorylation at Ser133 in
response to cAMP, calcium, stress, and mitogenic stimuli. Several kinases have
been reported to phosphorylate CREB at Ser133
(Mayr and Montminy, 2001).
In this study, we examined the mechanism by which PGI2 inhibits aortic smooth muscle cell proliferation and G1-S phase progression. We report that the PGI2 mimetic cicaprost inhibits cyclin A expression in primary murine aortic smooth muscle cells and established A10 rat aortic smooth muscle cells. Cicaprost treatment blocks the activation of cyclin E-cdk2, but this effect in itself is insufficient to explain the inhibition of cyclin A gene expression. Cicaprost also inhibits the binding of CREB and phospho-CREB to the CRE element in the cyclin A promoter. The inhibitory effects of cicaprost on cyclin E-cdk2 activity, CRE occupancy, and S phase entry are reversed in the presence of pertussis toxin. Overall, our results show that PGI2 inhibits the proliferation of aortic smooth muscle cells by coordinately blocking pocket protein- and CRE-dependent cyclin A gene expression, and that the underlying mechanism involves Gi signaling.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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)
and maintained in 1:1 Dulbecco's modified Eagle's/Ham's F-12 media
supplemented with 10% FBS and 2 mM L-glutamine. Experiments were
performed on cells at passage 3; these cultures were 
95% positive for
smooth muscle actin as determined by immunofluorescence microscopy (not
shown). The A10 rat aortic smooth muscle cell line (American Type Culture
Collection, Manassas, VA) was maintained in the same medium. In cell cycle
experiments, near-confluent monolayers of primary or A10 smooth muscle cells
were G0-synchronized by incubation in a serum-free defined media
(1:1 Dulbecco's modified Eagle's/Ham's F-12 media, 15 mM HEPES, pH 7.4, 3 mM
histidine, 4 mM glutamine, 8 mM sodium bicarbonate, 10 µM ethanolamine, 10
µg/ml transferrin, 0.1 µM sodium selenite, 0.1 µM MgCl2,
and 2 mg/ml heat-inactivated fatty acid-free bovine serum albumin) for 48 h
before stimulation with 10% FBS in the absence or presence of cicaprost.
Cicaprost was obtained and used under an agreement with Schering AG (Berlin,
Germany). In some experiments, cells were pretreated with 100 ng/ml of
pertussis toxin (List Biological Laboratories, Campbell, CA) for the last 16 h
of the 48 h serum starvation. Pertussis toxin was removed by washing; the
cells were then either directly stimulated with 10% FBS ± cicaprost or
trypsinized and replated in the presence of 10% FBS ± cicaprost. The
viability of cicaprost- or pertussis toxin-treated cells was approximately 95%
by trypan blue exclusion staining.
Immunofluorescence. Quiescent aortic smooth muscle cells and A10
smooth muscle cells (2 x 105 cells) were added to 35-mm
dishes containing autoclaved glass coverslips and incubated with 2 ml of
medium in 10% FBS in the absence or presence of 200 nM cicaprost. Cells were
fixed, permeabilized, and stained as described previously
(Roovers et al., 1999). For
detection of cyclin A, the cells were incubated for 1 h with ammonium
sulfate-fractionated rabbit polyclonal antibody against cyclin A (1:100
dilution) and then for 1 h with FITC-conjugated donkey anti-rabbit antibody
(1:100 dilution; Jackson Immunoresearch Labs, West Grove, PA). To monitor
entry into S phase, the incubation of primary and A10 smooth muscle cells with
10% FBS was performed in the presence of BrdU (3 µg/ml; Amersham,
Piscataway, NJ). Fixed cells were first incubated for 1 h with sheep anti-BrdU
(1:500 dilution; Biodesign, Saco, ME) and fresh DNase (280 units/ml) and then
for 1 h with FITC-conjugated donkey anti-sheep antibody (1:200 dilution;
Jackson Immunoresearch Labs) in PBS with 2% bovine serum albumin. Cell nuclei
were stained with 4,6-diamidino-2-phenylindole (2 µg/ml in PBS; Sigma,
Saint Louis, MO). The percentage of BrdU positive cells was assessed by
counting the number of FITC-stained cells relative to
4,6-diamidino-2-phenylindole—stained nuclei using epifluorescence
microscopy (100–200 cells in three or four fields of view were counted
for each time point).
Measurement of cAMP Levels. Cells were seeded in 12-well plates and cultured until they reached 90% confluence. The cells were pretreated with 3 µM indomethacin (Sigma) for 16 h before stimulation. On the day of the assay, cells were treated with 1 mM IBMX, a phosphodiesterase inhibitor, for 15 min, followed by stimulation with either increasing doses of cicaprost or increasing doses of forskolin (Sigma), in the absence or presence of 200 nM cicaprost, for 10 min. Cellular cAMP was extracted in 65% ethanol and quantified by radioimmunoassay according to the manufacturer's instructions (Amersham TRK432).
Western Blotting. Quiescent A10 smooth muscle cells were
trypsinized, replated at subconfluence (106 cells in 10 ml medium
per 100-mm culture dish), and incubated in 10% FBS in the absence or presence
of 200 nM cicaprost. Collected cells were lysed, and total protein
concentration was determined by Coomassie binding (Bio-Rad, Hercules, CA).
Equal amounts of protein (80 µg) were fractionated on reducing 12%
acrylamide gels and analyzed by Western blotting as described previously
(Roovers et al., 1999) using
antibodies specific for the following proteins: pRb (Zymed Labs, San
Francisco, CA), p107 (Santa Cruz Biotechnology, Santa Cruz, CA), cdk4 (Santa
Cruz Biotechnology or BioSource, Camarillo, CA), cyclin D1 (Upstate
Biotechnology, Lake Placid, NY), cyclin E (Santa Cruz Biotechnology), and cdk2
(Upstate Biotechnology). Rabbit polyclonal cyclin A antibody was prepared in
this laboratory using recombinant cyclin A as an immunogen.
In Vitro Kinase Assays. Quiescent A10 smooth muscle cells were
trypsinized, replated at subconfluence (106 cells in 10 ml of
medium per 100-mm culture dish), and incubated in 10% FBS in the absence or
presence of 200 nM cicaprost. Cyclin D1-cdk4 kinase assays were performed as
described previously (Welsh et al.,
2001) using 200 µg of cell lysates and glutathione
S-transferase-Rb as a substrate (Santa Cruz Biotechnology). The
reaction mixtures were fractionated on reducing SDS-gels (12% acrylamide),
electrophoretically transferred to nitrocellulose, and analyzed by
autoradiography. Nitrocellulose membranes were also Western blotted using
antibodies specific for cdk4 (BioSource) to assess the uniformity of the
immunoprecipitation. Cyclin E-cdk2 kinase assays were performed as described
previously (Zhu et al., 1996)
using 200 µg of cell lysate, 5 µg of anti-cyclin E (Santa Cruz
Biotechnology) and a 2-h incubation at 4°C with both primary antibody and
protein A-agarose. The collected immunoprecipitates were washed once with
lysis buffer and four times with ice-cold kinase buffer (50 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5
mM sodium fluoride, and 10 mM sodium orthovanadate). The washed
immunoprecipitate was resuspended in 50 µl of kinase buffer containing 5
µg of histone H1 (Upstate Biotechnology), 20 µM ATP, and 10 µCi of
[
-32P]ATP (3000 Ci/mmol). The kinase reaction was incubated
for 30 min at room temperature and stopped by the addition of 2x SDS
sample buffer and heating the sample for 2 min at 95°C. The reaction
products were analyzed by SDS gel electrophoresis, autoradiography, and
immunoblotting with anti-cdk2 (Upstate Biotechnology) as described above.
Kinase activity was quantified by PhosphorImager analysis and ImageQuant
software (Amersham Biosciences).
Electrophoretic Mobility Shift Assays. Nuclear extracts and
electrophoretic mobility shift assays (EMSAs) were prepared as described
previously (Bottazzi et al.,
2001). Double-stranded oligonucleotides that were used in these
studies contained either the wild-type cyclin A CRE
(5'-TGAATGACGTCAAGGCCGCGAG-3') or wild-type CCAAT element
(5'-CGAGCGCTTTCATTGGTCCATTTC-3'). For supershifts, the nuclear
extracts were preincubated (30 min at room temperature) with 5 µl of
antibodies against CREB, ATF-1, ATF-2, c-Jun, and c-Fos (all from Santa Cruz
Biotechnology) or phospho-CREB (Upstate Biotechnology) before the addition of
the radioactive probe. Protein-DNA complexes were fractionated on a 5%
polyacrylamide gel in 90 mM Tris, 90 mM Boric acid, 20 mM EDTA, pH 8.3, at 175
V for 3 h at 4°C. The gels were dried and analyzed by autoradiography.
Expression Vectors and Luciferase Constructs. The human cyclin A
promoter-firefly luciferase constructs used here were described previously
(Kramer et al., 1996). The
p434/cyclin A promoter construct contains 434 bases spanning from -270 to +164
of the cyclin A promoter upstream of the luciferase reporter. The p284/cyclin
A promoter construct (called +CRE) contains 284 bases from -120 to +164 of the
cyclin A promoter. Sequences upstream of the CRE have been deleted in +CRE.
The p225/cyclin A promoter construct (called -CRE) contains 225 bases from -61
to +164 of the cyclin A promoter and is a construct in which the CRE site from
the core promoter region, as well as all sequences upstream of the CRE have
been deleted. The human papilloma virus-type 18 E7 expression vector was the
generous gift of Lou Laimins (Northwestern University, Chicago, IL).
Cell Transfections and Promoter-Luciferase Assays. Transient
transfections of primary aortic and A10 smooth muscle cells with
promoter-luciferase vectors were performed using LipofectAMINE Plus
(Invitrogen, Carlsbad, CA) as described previously
(Bottazzi et al., 1999) using
2 x 105 cells, 1 µg of cyclin A promoter-luciferase
plasmid(s), 1 µg of the human papilloma virus-type 18 E7 expression vector
(or empty vector), and 0.1 µg of a Renilla reniformis luciferase
expression plasmid (pRL-CMV; Promega, Madison, WI) to control for transfection
efficiency. The transfection efficiency for both cell types was approximately
25% as determined by transient transfection of an enhanced green fluorescent
protein expression vector. The final amount of DNA transfected was brought to
2.1 µg for all samples by addition of the empty vector. After an overnight
recovery, the cells were serum-starved for 1 day in Dulbecco's modified
Eagle's medium and 1 mg/ml heat-inactivated fatty acid-free bovine serum
albumin. The serum-starved transfectants were directly stimulated with 10%
FBS-Dulbecco's modified Eagle's medium in the absence or presence of 200 nM
cicaprost or 10 ng/ml transforming growth factor- (TGF-). Cells
were washed with PBS, collected, lysed, and analyzed for luciferase and R.
reniformis luciferase activity using the Dual-Luciferase reporter assay
system (Promega, Madison, WI). Cyclin A promoter-driven luciferase activity
was then normalized to a constant activity of R. reniformis
luciferase to correct for variations in transfection efficiency.
Statistical Analysis. Data are presented as mean ± S.E.M. Statistical analyses were carried out using one-tailed t test. Differences were considered significant at p < 0.01.
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Results |
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10 nM in Chinese hamster
ovary cells expressing the mouse IP) compared with other prostanoid receptors
such as EP1 (Ki 1.3 µM) and
EP3 (Ki 170 nM)
(Narumiya et al., 1999). We
first used BrdU labeling to assess the effects of cicaprost on S-phase entry
in aortic smooth muscle cells. Primary aortic smooth muscle cells were
serum-starved and then stimulated with serum at subconfluence in the presence
of BrdU. The results showed that a majority of the primary aortic smooth
muscle cells entered S phase within 48 h of serum stimulation and that
cicaprost strongly inhibited S-phase entry
(Fig. 1A). Dose response curves
showed that cicaprost caused the expected increase in intracellular cAMP
levels in smooth muscle cells (EC50 40 nM;
Fig. 1B, IP +/+), and that
similar concentrations of cicaprost inhibited progression through
G1 phase (IC50 50 nM;
Fig. 1C, IP +/+). The
EC50 is slightly higher than others
(Kam et al., 2001) have
reported for cAMP production (EC50, 5–10 nM), perhaps because
we have studied endogenous IP rather than overexpressed IP. The effects of
cicaprost on both cAMP production and G1 phase progression were
lost in primary aortic smooth muscle cells from IP -/- mice
(Fig. 1, B and C). Cicaprost
also inhibited activation of the cyclin A promoter in aortic smooth muscle
cells transiently transfected with a cyclin A promoter-luciferase expression
vector (Fig. 2A) and blocked
the induction of cyclin A protein as determined by immunofluorescence
(Fig. 2B).
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Cicaprost Inhibits Pocket Protein Phosphorylation and Cyclin E-cdk2 Activity in Aortic Smooth Muscle Cells. To confirm the effects observed with primary smooth muscle cells and develop a system amenable to a mechanistic analysis, we examined the effect of cicaprost on G1 phase cell cycle progression in the A10 rat aortic smooth muscle cell line. Cicaprost inhibited S phase entry in these cells (Fig. 3A), and the IC50 (50 nM ± 2, n = 3, p < 0.01) was similar to that of the primary cells (see above). Cicaprost also inhibited activation of the cyclin A promoter (Fig. 3B) and induction of cyclin A protein (Fig. 3B, inset) in A10 smooth muscle cells.
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We then used A10 smooth muscle cells to determine the effect of cicaprost
on the G1 phase cyclin-cdks. Cicaprost did not affect the
expression of cyclin D1 (Fig.
4A), cdk4 (Fig.
4A), or cyclin D1-cdk4 activity
(Fig. 4B). The lack of effect
on cyclin D1-cdk4 was unexpected because the induction of cyclin D1 is
commonly blocked when cells arrest in G1 phase. However, this
result was supported by other experiments in A10 cells (data not shown), which
demonstrated that cicaprost had no effect on mitogen-activated protein kinase
kinase/extracellular signal-regulated kinase or phosphatidyl inositol-3
kinase/AKT signaling, the major pathways that regulate cyclin D1 expression
(Diehl et al., 1998;
Roovers and Assoian, 2000).
Although cicaprost was also without effect on the levels of cyclin E or cdk2
(Fig. 4C), it strongly
inhibited the activation of cyclin E-cdk2
(Fig. 4D). Curiously, cicaprost
did not increase the expression of p21Cip1, nor
did it lead to a consistent increase in the expression of
p27Kip1 (not shown). Therefore, the mechanism
by which cicaprost inhibits cyclin E-cdk2 activity may be complex and involve
coordinate effects on p27Kip1, cdk activating
kinase, and cdc25A (see Discussion). Consistent with the strong
inhibition of cyclin E-cdk2 activity, cicaprost blocked the
hyperphosphorylation of both pRb and p107 (shown by the absence of the slower
migrating form of these proteins; Fig.
4E).
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Inhibition of Pocket Protein Phosphorylation Cannot Fully Account for
the Inhibitory Effect of Cicaprost on the Cyclin A Promoter. Given the
effect of cicaprost on cyclin E-cdk2 activity and pRb and p107
phosphorylation, we considered the possibility that cicaprost blocks cyclin A
promoter activity by interfering with pocket protein function. Primary and A10
smooth muscle cells were cotransfected with the p434/cyclin A
promoter-luciferase reporter construct and a human papilloma virus-type 18 E7
expression vector [which sequesters pocket proteins and mimics the effect of
cdk-mediated phosphorylation (Vousden,
1993)]. Quiescent transfectants were stimulated with serum for 18
h before analysis of cyclin A promoter activity. The expression of E7 caused
cyclin A promoter activity to increase 5-fold, presumably because of enhanced
pocket protein sequestration and E2F release
(Fig. 5, 
). Cicaprost
inhibited activation of the cyclin A promoter, and E7 rescued promoter
activity partially, but not completely, in both primary and A10 smooth muscle
cells treated with cicaprost (Fig.
5, 
). The incomplete rescue was not caused by a partial
effect of E7, because inhibition of cyclin A gene expression by TGF-
[which occurs typically through a pocket protein-dependent mechanism
(Reynisdottir et al., 1995)]
was completely overcome by expression of E7
(Fig. 5, 
). Because
cicaprost inhibited the cyclin A promoter despite pocket protein sequestration
by E7, we reasoned that it was regulating cyclin A expression via a pocket
protein-independent as well as -dependent pathway.
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Cicaprost Inhibits Cre-Dependent Cyclin A Gene Expression. To address potential effects on pocket protein-independent cyclin A gene expression, we examined the effect of cicaprost on occupancy of the CCAAT or CRE sites in the cyclin A promoter. EMSAs showed that cicaprost did not inhibit occupancy of the CCAAT site but that it decreased occupancy of the CRE (Fig. 6A). Supershift EMSAs showed that CREB and phospho-CREB bind to the cyclin A CRE (Fig. 6B). A smaller amount of ATF-1 (relative to CREB) was also observed, but the binding of other potential CRE-binding proteins, such as ATF-2, c-fos, and c-jun, was undetectable (Fig. 6B). These results raised the possibility that cicaprost-dependent inhibition of CRE occupancy could account for the pocket-protein independent effect of cicaprost on cyclin A gene expression.
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Because IP signaling stimulates cAMP production, we expected that it might
stimulate the phosphorylation of CREB at Ser133 rather than block CRE
occupancy. Cicaprost did indeed stimulate CREB phosphorylation at Ser133, but
the effect was transient (30 min) and insufficient to sustain CREB
phosphorylation for the several hours needed to induce the cyclin A gene (data
not shown). In fact, under our experimental conditions (cells treated with
serum ± cicaprost), our data (Fig.
6B) show that serum alone supports CREB phosphorylation in late
G1 phase.
Complementary Effects of Cicaprost on Pocket Protein and CRE-Dependent Cyclin A Gene Expression in Aortic Smooth Muscle Cells. To assess the composite effects of cicaprost on CRE- and pocket protein-dependent cyclin A gene expression, we transiently cotransfected both primary and A10 smooth muscle cells with the E7 expression vector and the cyclin A promoter-luciferase reporter constructs, "+CRE" (Fig. 7A) and "-CRE" (Fig. 7B). Quiescent transfectants were stimulated with serum in the presence or absence of cicaprost. In the absence of E7, cicaprost inhibited luciferase activity whether or not the cyclin A promoter contained the CRE. This result supports our data showing that cicaprost inhibits cyclin E-cdk2 activity and pocket protein phosphorylation (Fig. 4, D and E). However, in the presence of E7, cicaprost inhibited luciferase activity only when the promoter contained the CRE. This result, which supports our finding that cicaprost inhibits CRE occupancy (Fig. 6A), demonstrates that the combined effects of cicaprost on pocket protein- and CRE-dependent transcription can fully account for its inhibitory effect on the cyclin A promoter.
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Inhibition of Gi Reverses the Inhibitory Effects of
Cicaprost. Activation of IP by cicaprost rapidly increases levels of cAMP
(Fig. 1B), and 8-bromo-cAMP
inhibits S-phase entry of A10 smooth muscle cells (not shown). These results
suggest a role for Gs in mediating the antimitogenic effect of
cicaprost but others have reported that the murine IP can also couple to the
heterotrimeric protein Gi
(Schwaner et al., 1995;
Lawler et al., 2001). We found
that although cicaprost stimulated cAMP levels in A10 smooth muscle cells
(2.70 ± 0.40-fold; mean ± S.E.M.), it reduced cAMP levels in
forskolin-stimulated cells (Fig.
8A), consistent with an IP-dependent activation of Gi
as well as Gs.
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We then sought to determine whether Gi-mediated signaling was
involved in the inhibitory effects of cicaprost on cell cycle progression.
Serum-stimulated primary and A10 smooth muscle cells were incubated with
pertussis toxin, an irreversible inhibitor of Gi proteins. Neither
cicaprost nor pertussis toxin affected CCAAT occupancy
(Fig. 8B, lanes 1–3).
However, pertussis toxin reversed the inhibitory effect of cicaprost on CRE
occupancy (Fig. 8B, compare
lanes 5 and 7), and the complexes seen in the cicaprost/pertussis
toxin-treated cells contained CREB and phospho-CREB
(Fig. 8B, lanes 12 and 13),
just as the complexes did in the absence of cicaprost
(Fig. 8B, lanes 8–11).
Furthermore, the inhibitory effects of cicaprost on cyclin E-cdk2 activity
(Fig. 8C), cyclin A induction
(Fig. 8D), and S phase entry
(Fig. 8E) were also reversed by
pertussis toxin. This was not a generic effect because pertussis toxin did not
affect the extent of S phase entry in response to serum
(Fig. 8E) or reverse
TGF--mediated inhibition of smooth muscle cell cycle progression (data
not shown). Thus, Gi signaling is required for the antimitogenic
effects of cicaprost in smooth muscle cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Although we see a nearly complete inhibition of cyclin E-cdk2 activity by
cicaprost in A10 smooth muscle cells, the mechanism by which this occurs is
not clear. Cicaprost neither increased the expression of
p21Cip1 nor consistently increased
p27Kip1 levels. Although cdk2 activity is
typically regulated by changes in the expression of these cdk inhibitors,
others have reported that cdc25A and perhaps cdk-activating kinase may also
regulate cyclin E-cdk2 activity (Nagahara
et al., 1999). Similarly, SPARC, a matrix-associated glycoprotein,
inhibits cyclin E-cdk2 activity in human arterial smooth muscle cells without
affecting p21Cip1,
p27Kip1, cyclin E, or cdk2
(Motamed et al., 2002).
PGI2 signals through its interaction with the IP receptor, a
heterotrimeric G-protein-coupled receptor
(Coleman et al., 1994). It is
well established that IP couples to Gs
(Namba et al., 1994) and
increases cAMP production. Thus, PGI2 would be expected to
stimulate the phosphorylation of CREB (a known cAMP-dependent kinase
substrate). Although we could detect a rapid increase in CREB phosphorylation
at Ser133 in cicaprost-treated smooth muscle cells, the effect was very
transient (lasting 30 min) and insufficient to sustain CREB
phosphorylation for the several hours needed to induce the cyclin A gene.
However, EMSAs performed at 18 h (when the cyclin A promoter is active) showed
that cicaprost decreases the occupancy of the cyclin A CRE in serum-stimulated
A10 smooth muscle cells, indicating that cicaprost is blocking the binding of
CREB and phospho-CREB to the CRE. CREB can also be phosphorylated at Ser142 by
casein kinase II and calcium-calmodulin kinase II, and this phosphorylation
blocks the formation of CREB-CBP complexes and the activation of target genes
(Sun et al., 1994). The
particular kinases required for CREB phosphorylation at Ser133 and Ser142 in
our system, and the mechanism of the IP effect is currently being
investigated.
Interestingly, we find that all of the antimitogenic effects of cicaprost
(i.e., the inhibition of CRE occupancy, inhibition of cyclin E-cdk2 activity,
inhibition of cyclin A gene expression, and inhibition of S-phase entry) are
reversed in cells treated with pertussis toxin. Thus, in both primary and A10
smooth muscle cells, the antimitogenic effect of cicaprost requires
Gi signaling. Although it is possible that Gi acts alone
to mediate the antimitogenic effect of cicaprost on cyclin A expression and
G1 phase progression, the antimitogenic effect of 8-bromo-cAMP
suggests a role for Gs signaling. It is possible that Gs
and Gi must both be activated, either sequentially (e.g., as
reported by Lawler et al.,
2001) or in parallel. Future studies will be directed at
characterizing the relative roles of Gi and Gs in the
antimitogenic effects of cicaprost.
Together with our previous studies on regulation of the cyclin A gene
(Bottazzi et al., 2001), the
studies described here lead to a working model for the inhibitory effect of
PGI2 on the cyclin A promoter and, consequently, the proliferation
of aortic smooth muscle cells (Fig.
9). We have previously reported that serum-derived mitogens
stimulate the phosphorylation of CREB
(Bottazzi et al., 1999), and we
now show that PGI2 inhibits CRE occupancy by CREB and phospho-CREB.
Mitogens and the extracellular matrix also act coordinately to activate cyclin
D1-cdk4 and cyclin E-cdk2, but PGI2 specifically inhibits the
activation of cyclin E-cdk2, thereby preventing pocket protein inactivation.
We propose that by coordinately regulating CRE- and pocket protein-dependent
cyclin A gene expression, PGI2 inhibits entry into S phase and
prevents the proliferation of aortic smooth muscle cells. These effects are
likely to underlie the antiproliferative effect of PGI2 and IP in
injury-induced cardiovascular disease.
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Acknowledgements |
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Footnotes |
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D.K. and S.A.S. contributed equally to this work.
ABBREVIATIONS: PGI2, prostacyclin; IP, prostacylin receptor; cdk, cyclin-dependent kinase; pRb, retinoblastoma protein; CRE, cAMP response element; CDE, cell cycle-dependent element; CHR, cell cycle gene homology region; CREB, cAMP response element-binding protein; ATF, activating transcription factor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; BrdU, bromodeoxyuridine; PBS, phosphate-buffered saline; IBMX, 3-isobutyl-1-methylxanthine; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; TGF, transforming growth factor.
Address correspondence to: Dr. Richard K. Assoian, 167 Johnson Pavilion, 3620 Hamilton Walk, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084. Email: rka{at}pharm.med.upenn.edu
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) and IP -/- (
) mice pretreated with IBMX and then stimulated with
increasing doses of cicaprost for 10 min. Cells were collected, and cAMP
levels were quantified by radioimmunoassay. Results show the mean ±
S.E.M., n = 3; **, p < 0.01. C, quiescent
primary aortic smooth muscle cells [IP +/+ (
, p < 0.01 compared with
E7-transfected cells stimulated with FBS.
