© 2001 American Heart Association, Inc.
Cellular Biology |
Membrane-Bound Protein Kinase A Inhibits Smooth Muscle Cell Proliferation In Vitro and In Vivo by Amplifying cAMP–Protein Kinase A Signals
From the Division of Cardiology (C.I.) and Dipartimento di Medicina Sperimentale e Clinica (E.V.A.), "Magna Graecia" University, Catanzaro, and Division of Cardiology (E.S., C.C., C.P., L.C., D.T., E.D.L., M.C.) and Dipartimento di Biologia e Patologia Molecolare e Cellulare (A.G., G.A., A.F., E.V.A.), Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, University Federico II, Naples, Italy.
Correspondence to Enrico V. Avvedimento or Ciro Indolfi, Department of Patologia Molecolare e Cellulare, Centro di Endocrinologia ed Oncologia Sperimentale, CNR, Via S. Pansini, 5 80131, Naples, Italy. E-mail avvedim{at}unina.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—cAMP-dependent protein kinase is anchored to discrete cellular compartments by a family of proteins, the A-kinase anchor proteins (AKAPs). We have investigated in vivo and in vitro the biological effects of the expression of a prototypic member of the family, AKAP75, on smooth muscle cells. In vitro expression of AKAP75 in smooth muscle cells stimulated cAMP-induced transcription, increased the levels of the cyclin-dependent kinase-2 inhibitor p27kip1, and reduced cell proliferation. In vivo expression of exogenous AKAP75 in common carotid arteries, subjected to balloon injury, significantly increased the levels of p27kip1 and inhibited neointimal hyperplasia. Both the effects in smooth muscle cells in vitro and in carotid arteries in vivo were specifically dependent on the amplification of cAMP-dependent protein kinase (PKA) signals by membrane-bound PKA, as indicated by selective loss of the AKAP75 biological effects in mutants defective in the PKA anchor domain or by suppression of AKAP effects by the PKA-specific protein kinase inhibitor. These data indicate that AKAP proteins selectively amplify cAMP-PKA signaling in vitro and in vivo and suggest a possible target for the inhibition of the neointimal hyperplasia after vascular injury.
Key Words: A-kinase anchor proteins • p27 • protein kinase A • smooth muscle cells
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cyclic AMP controls growth and differentiation in a variety of organisms and cell types.1 2 In eukaryotes, cAMP binds the regulatory subunit of cAMP-dependent protein kinases (PKAs). This releases the catalytic subunit (C-PKA), which phosphorylates a wide variety of substrate proteins. A fraction of the C-PKA migrates to the nucleus and phosphorylates nuclear proteins and transacting factors.3
PKA is targeted to certain subcellular locations by specific anchor proteins (A-kinase anchor proteins, AKAPs). Localized PKA holoenzymes might in vivo perform important aspects of cAMP-activated signal transduction. We have previously provided evidence that links membrane targeting of PKAII to cAMP-dependent gene transcription in differentiated and nondifferentiated cells.4 5 In thyroid, neuronal, and kidney cells, displacement of immobilized PKAII from perinuclear sites to the cytoplasm impaired cAMP-induced transcription.4 Conversely, overexpression of AKAP75, a prototype AKAP that targets PKA to the membranes, enhanced the propagation of cAMP signals to the nucleus.5
However, the biological effects of AKAPs on smooth muscle cell (SMC) growth in vitro and in vivo are not known. Therefore, we performed 2 experimental models to assess the effects of AKAP expression on SMC proliferation both in vitro and in vivo. In the first model, we have transfected stabilized SMCs derived from aorta with expression vectors encoding the prototypic member of AKAP family, AKAP75 and its derivatives. The same vectors were also used in vivo in the rat model of angioplasty. After balloon injury, the cells in the media of the arterial wall are potently stimulated and proliferate vigorously, thus generating the so-called neointima.6 This system is ideally suited for assessing nontransformed SMC proliferation in vivo7 8 9 10 and testing innovative strategies to prevent vascular proliferative disorders such as atherosclerosis and restenosis.
Here we report that overexpression in vitro and in vivo of AKAP75 potently inhibits SMC proliferation and prevents neointima formation after balloon injury. These effects are specifically dependent on the amplification of cAMP signals by membrane-bound PKA and suggest a potential target site for therapeutic control of balloon angioplasty–induced neointimal formation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DNA Plasmids, Cell Culture, and Transfections
The plasmids used in this study are (1) cAMP-responsive element (CRE)-chloramphenicol acetyltransferase (CAT), carrying the Escherichia coli CAT gene under the control of the somatostatin promoter11 ; (2) CMV-AKAP75, containing the bovine brain RIIß anchor protein under the control of cytomegalovirus (CMV) promoter12 ; (3) CMV-AKAP45, a truncated mutant of AKAP75 and a derivative of AKAP45 mutated in the RII binding site (AKAP45mut)12 ; (4) RSV-LacZ, the E. coli LacZ gene driven by the long terminal repeats of Rous sarcoma virus (RSV)13 ; (5) RSV-PKI,14 containing the RSV promoter driving the expression of the rabbit PKA inhibitor gene; (6) PKImut, the rabbit PKA inhibitor single-point mutant gene14 ; (7) RSV-Neo, the neomycin resistance gene; and (8) RSV-CAT, which carries the CAT gene under the control of RSV promoter.
Rat aortic embryonal SMCs (A10 cells) were cultured in DMEM (10% FCS), and DNA transfections were carried out by the calcium phosphate procedure.11 For stable transformed lines (CMV-AKAP75, RSV-Neo, and AKAP45mut), after 24 hours of incubation in nonselective medium, the cells were plated in the selective medium with a neomycin analogue (G418, 500 µg/mL).
CAT Assay
Dishes were transfected with 5 µg each of CRE-CAT,
RSV-Lac Z, AKAP75, AKAP45, AKAP45mut, and
protein kinase inhibitor (PKI), as well as up to 21 µg of salmon
sperm DNA.4 CAT activity was
performed 48 hours after transfection before and after 4 hours of
8-bromo-cAMP (8-Br-cAMP)
incubation.11
SMC Cultures
To assess the effects of AKAP75 transfection on SMC
proliferation in vitro, the cultures were synchronized by starvation
for 48 hours in serum-free DMEM and then serum-restimulated in the
presence of [3H]thymidine. DNA synthesis
cells were measured by [3H]thymidine
incorporation as described
previously.12 Experiments
were performed in quadruplicate, and the data shown are a mean of 4
independent experiments that all gave similar
results.
Western Immunoblot Analysis
Dishes were transfected with 5 µg each of RSV-Lac
Z, AKAP75, and PKI. Western immunoblot analysis was performed 48 hours
after transfection before and after 4 hours of 8-Br-cAMP
incubation.12 In brief,
after cell homogenization, proteins were transferred to a
nitrocellulose membrane (Hybond-ECL, Amersham Life Sciences) by semidry
electroblotting for 1 hour. The membranes were blocked for 1 hour at
room temperature with Blotto-Tween (5% nonfat dry milk, 0.1%
Tween-20) and incubated with a murine polyclonal
p27kip1 IgG1 (0.25 mg/mL, dilution 1:1000,
Santa Cruz Biotechnology, Inc). Bound antibody was detected with
horseradish peroxidase–labeled rabbit anti-mouse IgG conjugate
(Prosan, dilution 1:2000 in Blotto-Tween) and visualized by enhanced
chemiluminescence (Amersham).
Ligand Blot Analysis or Overlay
To identify AKAP75-RIIß complex, after
electrophoresis, the proteins were transferred to nitrocellulose
filters, probed with labeled RIIß (AKAP75 antibody was kindly
provided from Dr CS Rubin (Albert Einstein College of Medicine,
Bronx, NY), and identified by autoradiography as previously
described.11
Animal Preparation
All animals received humane care in accordance with
the animal use principles of the American Society of Physiology. In 64
Wistar rats (350 to 400 g), balloon injury of the common carotid
artery was performed as previously
described.7 8 9
Site-specific gene transfer was achieved by transfecting arteries with
the use of a DNA/gel (30% wt/vol) solution (pluronic gel F127, BASF
Corp) containing different plasmids (200 µg each plasmid), applied on
the artery immediately after the
injury.8 9
We assessed the effects of balloon injury (n=11) alone and of pluronic gel application (without plasmids) (n=6) after the injury, on neointimal formation 14 days after injury.11 Then we determined the effect of the following plasmid constructs: (1) RSV–ß-Gal (n=11); (2) CMV-AKAP75 (n=12); (3) CMV-AKAP45 (n=7); and (4) finally, the effect of simultaneous transfection of AKAP75 with PKI15 (n=5) or PKImut (n=4).
Localization of AKAP75 and
p27kip1 in the Vessel Wall
Two days after balloon injury, additional animals
from the control and AKAP75 groups were euthanized by administration of
an overdose of pentobarbital, and the arteries were perfusion-fixed in
4% formaldehyde.16 Arteries
were divided into 2-mm-thick segments, overlaid with OCT compound, and
frozen in liquid nitrogen. Cryostat sections (7 µm) were mounted on
poly-L-lysine–coated
slides. To detect AKAP75 and p27kip1
immunoreactivity, sections were washed twice with Tris-buffered saline
(TBS), blocked with rabbit serum, diluted 1:5 in TBS for 45 minutes,
incubated overnight with polyclonal murine anti-AKAP75 or
anti-p27kip1 IgG1 antibody (2 µg/mL, Santa
Cruz Biotechnology, Inc), and incubated for 1 hour with a rabbit
anti-mouse IgG conjugated with horseradish peroxidase (dilution 1:50;
preabsorbed overnight at 4°C with 10% preimmune rat serum and 3%
BSA). Antibody binding in the vessel wall was visualized with
3,3'-diaminobenzidene (Sigma Chemical Co) in 0.1 mol/L Tris buffer (pH
7.2) containing 0.01%
H2O2. Sections were
counterstained with Harris hematoxylin, dehydrated, and mounted with
dePex mounting medium. The number of cells expressing
p27kip1 was quantified as the ratio of
nuclear-positive cells on total cells in each
field.
Morphology
At the time of the final experiment, the arteries
were dissected free from the surrounding tissues, and rats were
euthanized for structural and immunohistochemical
analyses.7 All data are
mean±SEM. ANOVA for repeated measures was performed using the SPSS
program, version 10.0; the Tukey test was used to compare single
mean
values.17
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We first determined whether the expression of AKAP75, a prototypic member of the AKAP family, influences cAMP signaling in cultured aortic SMCs, by measuring the transcriptional activity of a cAMP-inducible promoter driving the expression of the bacterial enzyme, CAT (CRE-CAT) (Figure 1
).
|
Expression of AKAP75 induced CRE-CAT in the absence of cAMP
stimulation
(Figure 1
). This effect was mediated by PKA, given that it
was reversed by the simultaneous transfection of the PKA nuclear
inhibitor PKI, but not by a single-point PKI mutant that did not
inhibit PKA (data not shown). These effects were specific to the CRE
promoter because RSV-CAT under the same conditions was not activated by
AKAP75 or cAMP
(Figure 1). Moreover, expression of AKAP45, a derivative of
AKAP75 carrying a deletion in the membrane-anchoring domain (residues 1
to 180; Reference 1212 ), did inhibit CRE-CAT both in the presence and the
absence of cAMP
(Figure 1). Because AKAP45 binds the endogenous PKA and
translocates it to the
cytosol,12 the inhibition of
CRE-CAT transcription is apparently dependent on the loss of cellular
membrane-bound PKA. Mutation of the PKA binding site in AKAP45
(A45mut) completely eliminated the negative
effect of AKAP45 on cAMP-induced transcription, indicating that both
the positive (AKAP75) and the negative (AKAP45) effects on CRE-CAT
transcription were mediated by the PKA binding domain of AKAP
(Figure 1). This finding also indicates the high degree of
specificity of AKAP75 in cAMP signal transduction, because AKAP75 binds
other signaling molecules, such as PKC and calcineurin, that might be
activated by
injury.18
To test the biological consequences of AKAP75 expression, we
analyzed the DNA synthesis in AKAP75-transfected cells.
Figure 2 shows that the DNA synthesis of AKAP75-expressing
cells was dramatically reduced. As control we used cells transfected
with AKAP45mut indicated
above.
|
Mock or RSV-NEO–expressing cells show a profile of DNA
synthesis and proliferation comparable with those of
AKAP45mut-expressing cells (data not shown).
Because levels of p27kip1, a specific
cyclin-dependent kinase-2 (cdk2) inhibitor, are stimulated by
cAMP,19 we determined
p27kip1 concentration in AKAP75-expressing
cells by Western blotting analysis.
Figure 3 shows that cells expressing AKAP75 contained higher
p27kip1 levels relative to control cells.
Moreover, these cells responded very efficiently to cAMP by increasing
p27 levels. PKI but not the mutated variant reduced cAMP-induced p27
levels in AKAP75-expressing cells. These data replicate the effects of
AKAP75 on CRE-CAT transcription and indicate that p27 levels are
tightly controlled by cAMP-PKA.
|
To translate these results in vivo, we determined the
biological consequences of AKAP75 expression in rat carotid arteries
(n=64), subjected to vascular injury as previously
described.7 Rat carotid
arteries subjected to balloon injury were "transfected" in vivo
with plasmid vectors expressing AKAP75, AKAP45, and PKI wild-type or
its mutated variant (PKImut). We first
determined, 48 hours after the injury, the expression of exogenous
AKAP75 gene in the arterial walls with antibodies specific to AKAP75.
Cross sections of the arterial walls showed the specific AKAP75 signal
in the SMCs of the tunica media of the vessel only in AKAP75-treated
animals
(Figure 4). The expression was maximal 48 hours after the
injury and steadily decreased thereafter (data not shown).
|
In parallel experiments, the animals were euthanized 14 days
after injury to determine the effects of AKAP75 expression on in vivo
SMC proliferation. Figure 6 shows that AKAP75 expression reduced
significantly the formation of neointima 14 days after arterial balloon
injury. DNA expressing vectors containing different viral (RSV-CMV) or
eukaryotic (transgenic) promoters driving the expression of
bacterial or animal proteins did not inhibit the formation of the
neointima.8
|
The arteries treated with AKAP75 in vivo were also stained
with p27 antibody.
Figure 5 shows that AKAP75-treated arteries contained a
significant fraction of cells that expressed
p27kip1 48 hours after the arterial injury
(33% versus 18% of positive cells per field,
P<0.05) and were not present
in arteries transfected with control vectors.
|
Figure 6 shows representative cross sections of each group
of animals studied. The inhibition of neointimal hyperplasia after
balloon injury produced by AKAP75 transfection was totally abolished by
the wild-type PKA-specific inhibitor
PKI14 but not by its mutated
version. The neointima area was 0.129±0.040
mm2 in the group transfected with AKAP75 and
PKI (n=5) compared with 0.079±0.017 mm2 in
the group treated with AKAP75 alone (n=12)
(P<0.01 versus AKAP75±PKI).
Similarly, the neointima/media ratio was 0.621±0.123 in AKAP75 treated
arteries compared with 1.118±0.316 in the group treated with AKAP75
and PKI (P<0.01 versus
AKAP75). On the other hand, after balloon injury in the group treated
with AKAP75 and PKI mutant, no difference was observed either in
neointimal area (n=4) (0.073±0.014 mm2;
P=NS versus AKAP75) or in
neointima/media ratio (0.717±0.142;
P=NS versus AKAP75) compared
with the AKAP75 group.
The AKAP75 mutant, AKAP45, carrying the deletion of the
membrane-anchoring domain, did not inhibit SMC proliferation in vitro
(Figure 2) nor the neointimal formation after balloon injury
(Figure 6). In the AKAP45-treated group (n=7), at 14 days
after balloon injury the neointimal area was 0.129±0.024
mm2
(P<0.01 versus AKAP75).
Similarly, the neointima/media ratio was 0.621±0.123
(P<0.01 versus AKAP75). These
findings indicate that AKAP75 effects are tightly dependent on the PKA
anchoring domain and that amplification of cAMP signaling reduces
neointimal hyperplasia after balloon injury.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The major findings of the present study are that AKAP75 expression in cultured SMCs in vitro and in arteries subjected to balloon injury amplifies and stimulates cAMP-PKA signaling resulting in activation of cAMP-induced transcription, increased levels of p27kip1, and suppression both of SMC growth in vitro and of neointimal hyperplasia after balloon injury.
These effects in vitro and in vivo were dependent on selective stimulation of AKAP-targeted PKAII. Indeed, a PKA-specific inhibitor, PKI, but not its inactive mutant, inhibits the effects of AKAP both in vivo and in vitro. AKAP75 binds and anchors the regulatory subunit of PKA to discrete subcellular membranes. Apparently, the membrane system where AKAP-PKA localizes can vary and depends on the cell type.5 20 21 We have shown that independently on the specific localization of the complex AKAP75-PKA in different cell types (kidney, thyroid, and neuronal cells), the invariant result of AKAP75 expression is the amplification of cAMP signaling to the nucleus. PKA in AKAP75-expressing cells appears to be more stable.5 11 12 Although we did not explore the specific localization of the complex AKAP-PKAII in SMCs, the net effect of AKAP75 expression in cultured SMCs in vitro and in arteries in vivo was the amplification of cAMP signaling.
The selective inhibition of AKAP75 effects by selected
mutants provides important information about the mechanism(s)
underlying the AKAP75 function. A mutant of AKAP75, AKAP45, which lacks
the targeting domain, does not prevent neointimal hyperplasia after
balloon injury, inhibits CRE-CAT transcription, and decreases
p27kip1. This finding indicates that the RII
binding domain is essential for the effects of AKAP75 on cAMP
signaling. Moreover, it suggests a function for the endogenous
membrane-bound PKA in SMCs. Thus, AKAP45 inhibits basal and
cAMP-stimulated transcription, whereas it has no effect on the
formation of neointima
(Figure 1 and
Figure 6). Because this mutant binds endogenous PKA and
translocates it to the
cytosol,11 12 the
inhibition of CRE-CAT transcription and the loss of growth-inhibitory
effect suggest that in SMCs endogenous membrane-bound PKA amplifies the
effects of cAMP signaling on transcription and inhibits proliferation.
We suggest that cytosolic PKA bound to AKAP45 is readily dissociated by
basal cAMP, and it is degraded faster. Cells expressing AKAP45 do not
transcribe efficiently cAMP-induced genes, contain less p27, and grow
faster.12 Furthermore, the
effects of AKAP45 are critically dependent on the interaction with PKA,
given that the derivative mutant in the RII binding site
(AKAP45mut) did not inhibit CRE-CAT
transcription or SMC growth
(Figures 1 and 2). These data further support the notion that
the effects of AKAP on neointima formation after balloon injury are
mediated by PKA. Interestingly, AKAP75 binds other signaling molecules,
such as PKC or calcineurin, which might independently be activated by
arterial balloon injury and might contribute to the regulation of
neointima formation as well. Our data suggest that the mechanism of SMC
growth inhibition in vivo and in vitro by AKAP75 is related to the
amplification of cAMP signals, which can inhibit transcription of
cyclin D1 expression15 and
increase p27 levels. We have data indicating that cAMP-PKA signals
influence the degradation and not the synthesis of
p27.12 Under these
conditions, even a small quantitative effect (
3-fold) on p27 levels
may have a dramatic effect on DNA synthesis, because p27 levels in
G1 are in equilibrium with cdk2, and any
variation in this ratio can trigger cdk2
activity.12
The present study further supports the hypothesis that ras pathway inhibition and cAMP stimulation are powerful means to inhibit neointimal formation after balloon injury.8 10 In conclusion, expression of AKAP75 in vitro stimulated cAMP-induced transcription, increased the levels of the cdk2 inhibitor p27kip1, and reduced SMC proliferation. In an in vivo model of vascular injury, AKAP75 significantly increased p27kip1 levels and inhibited neointimal hyperplasia. These effects on SMCs in vitro and on injured arteries in vivo were specifically dependent on the amplification of cAMP-PKA signals by membrane-bound PKA. These data indicate that AKAP proteins selectively amplify cAMP-PKA signaling in vitro and in vivo and support a possible target for the inhibition of the neointimal hyperplasia after vascular injury.
|
Acknowledgments |
|---|
This study was supported in part by Associazione Italiana per la Ricerca sul Cancro, Ministero dell’Universitá e della Ricerca Scientifica e Technologica, and Consiglio Nazionale delle Ricerche (targeted project, biotechnology). We thank Dr CS Rubin (Albert Einstein College of Medicine, Bronx, NY) for kindly providing anti-AKAP75 antibody.
|
Footnotes |
|---|
Original received January 18, 2000; resubmission received August 10, 2000; revised resubmission received December 15, 2000; accepted December 15, 2000.
1 Both authors contributed equally to this study. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- Grieco D, Porcellini A, Avvedimento VE, Gottesman ME. Requirement for cAMP-PKA pathway activation by M phase-promoting factor in the transition from mitosis to interphase. Science. 1996;271:1718–1722.[Abstract]
- Brandon EP, Idzerda RL, McKnight GS. PKA isoforms, neural pathways, and behaviour: making the connection. Curr Opin Neurobiol. 1997;3:397–403.
- Montminy M. Transcriptional regulation by cyclic AMP. Annu Rev Biochem. 1997;66:807–822.[Medline] [Order article via Infotrieve]
-
Cassano S, Gallo A,
Buccigrossi V, Porcellini A, Cerillo R, Gottesman ME, Avvedimento EV.
Membrane localization of cAMP-dependent protein kinase amplifies cAMP
signaling to the nucleus in PC12 cells.
J Biol Chem. 1996;271:29870–29875.
[Abstract/Free Full Text] - Feliciello A, Li Y, Avvedimento EV, Gottesman ME, Roubin CS. A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus. Curr Biol. 1997;7:1011–1014.[Medline] [Order article via Infotrieve]
- Clowes AW, Reidy MA, Clowes NM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–337.[Medline] [Order article via Infotrieve]
-
Indolfi C, Esposito
G, Rapacciuolo A, Di Lorenzo E, Feliciello A, Porcellini A, Avvedimento
VE, Condorelli M, Chiariello M. Smooth muscle cell proliferation is
proportional to the degree of balloon injury in a rat model of
angioplasty. Circulation. 1995;92:1230–1235.
[Abstract/Free Full Text] - Indolfi C, Avvedimento EV, Rapacciuolo A, Di Lorenzo E, Esposito G, Stabile E, Feliciello A, Mele E, Giuliano P, Condorelli G, Chiariello M. Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo. Nat Med. 1995;1:541–54.[Medline] [Order article via Infotrieve]
- Simons M, Edelman ER, DeKeyser JL, Langer, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992;359:67–70.[Medline] [Order article via Infotrieve]
- Indolfi C, Avvedimento EV, Di Lorenzo E, Esposito G, Rapacciuolo A, Giuliano P, Grieco D, Cavuto L, Stingone AM, Ciullo I, Condorelli G, Chiariello M. Activation of cAMP-PKA signaling inhibits smooth muscle proliferation induced by vascular injury. Nat Med. 1997;3:775–779.[Medline] [Order article via Infotrieve]
-
Cassano S, Di
Lieto A, Cerillo R, Avvedimento EV. Membrane-bound cAMP-dependent
protein kinase controls cAMP-induced differentiation in PC12 cells.
J Biol Chem. 1999;274:32574–32579.
[Abstract/Free Full Text] -
Feliciello A,
Gallo A, Mele E, Porcellini A, Troncone G, Garbi C, Gottesman ME,
Avvedimento EV. The localization and activity of cAMP-dependent protein
kinase affect cell cycle progression in thyroid cells.
J Biol Chem. 2000;275:303–311.
[Abstract/Free Full Text] -
Sassone-Corsi P,
Visvader J, Ferland L, Mellon PL, Verma IM. Induction of proto-oncogene
fos transcription through the
adenylate cyclase pathway: characterization of a cAMP-responsive
element. Genes Dev. 1988;2:1529–1538.
[Abstract/Free Full Text] -
Day RN, Walder
JA, Maurer RA. A protein kinase inhibitor gene reduces both basal and
multihormone-stimulated prolactin gene transcription.
J Biol Chem. 1989;264:431–436.
[Abstract/Free Full Text] - L’Allemain G, Lavoie JN, Rivard N, Baldin V, Pouyssegur J. Cyclin D1 expression is a major target of the cAMP-induced inhibition of cell cycle entry in fibroblasts. Oncogene. 1997;14:1981–1990.[Medline] [Order article via Infotrieve]
- Warnke R, Levy R. Detection of T and B cell antigens hybridoma monoclonal antibodies: a biotin-avidin-horseradish peroxidase method. J Histochem Cytochem. 1980;28:771–776.[Abstract]
- Dixon WJ, Massey J. Introduction to Statistical Analysis. New York: McGraw-Hill; 1969.
-
Skaletz-Rorowski
A, Waltenberger J, Muller JG, Pawlus E, Pinkernell K, Breithardt G.
Protein kinase C mediates basic fibroblast growth factor-induced
proliferation through mitogen-activated protein kinase in coronary
smooth muscle cells. Arterioscler Thromb
Vasc Biol. 1999;19:1608–1614.
[Abstract/Free Full Text] -
Sherr CJ, Roberts
JM. Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev. 1995;9:1149–1163.
[Free Full Text] -
Paolillo M,
Feliciello A, Porcellini A, Garbi C, Bifulco M, Schinelli S, Ventra C,
Stabile E, Ricciarelli G, Schettini G, Avvedimento VE. The type and
localization of cAMP signals to the nucleus in cortical and granule
cerebellar cells. J Biol
Chem. 1999;274:6546–6552.
[Abstract/Free Full Text] -
Feliciello A,
Giuliano P, Porcellini A, Garbi C, Obici S, Mele E, Angotti E, Grieco
D, Amabile G, Cassano S, Li Y, Musti AM, Rubin CS, Gottesman ME,
Avvedimento EV. The v-Ki-Ras oncogene alters cAMP nuclear signaling by
regulating the location and the expression of cAMP-dependent protein
kinase IIß. J Biol Chem. 1996;271:25350–25359.
[Abstract/Free Full Text]
This article has been cited by other articles:
 |
|
 |
|
  Z. Yin, G. N. Jones, W. H. Towns II, X. Zhang, E. D. Abel, P. F. Binkley, D. Jarjoura, and L. S. Kirschner Heart-Specific Ablation of Prkar1a Causes Failure of Heart Development and Myxomagenesis Circulation, March 18, 2008; 117(11): 1414 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Torella, A. Curcio, C. Gasparri, V. Galuppo, D. D. Serio, F. C. Surace, A. L. Cavaliere, A. Leone, C. Coppola, G. M. Ellison, et al. Fludarabine prevents smooth muscle proliferation in vitro and neointimal hyperplasia in vivo through specific inhibition of STAT-1 activation Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2935 - H2943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Streb and J. M. Miano Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin) J. Biol. Chem., July 29, 2005; 280(30): 28007 - 28014. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Sakaguchi, T. Asai, D. Belov, M. Okada, D. J. Pinsky, A. M. Schmidt, and Y. Naka Influence of ischemic injury on vein graft remodeling: Role of cyclic adenosine monophosphate second messenger pathway in enhanced vein graft preservation J. Thorac. Cardiovasc. Surg., January 1, 2005; 129(1): 129 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Chotani, S. Mitra, A. H. Eid, S. A. Han, and N. A. Flavahan Distinct cAMP signaling pathways differentially regulate {alpha}2C-adrenoceptor expression: role in serum induction in human arteriolar smooth muscle cells Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H69 - H76. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Torella, D. Leosco, C. Indolfi, A. Curcio, C. Coppola, G. M. Ellison, V. G. Russo, M. Torella, G. L. Volti, F. Rengo, et al. Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2850 - H2860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Stabile, Y. F. Zhou, M. Saji, M. Castagna, M. Shou, T. D. Kinnaird, R. Baffour, M. D. Ringel, S. E. Epstein, and S. Fuchs Akt Controls Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo by Delaying G1/S Exit Circ. Res., November 28, 2003; 93(11): 1059 - 1065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Indolfi, D. Torella, C. Coppola, A. Curcio, F. Rodriguez, A. Bilancio, A. Leccia, O. Arcucci, M. Falco, D. Leosco, et al. Physical Training Increases eNOS Vascular Expression and Activity and Reduces Restenosis After Balloon Angioplasty or Arterial Stenting in Rats Circ. Res., December 13, 2002; 91(12): 1190 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Indolfi, D. Torella, C. Coppola, E. Stabile, G. Esposito, A. Curcio, A. Pisani, L. Cavuto, O. Arcucci, M. Cireddu, et al. Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H760 - H767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Indolfi Genetic factors in atherosclerosis: status and perspectives Eur. Heart J. Suppl., March 1, 2002; 4(suppl_B): B14 - B16. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||