Vol. 7, 1453-1460,
November
Cell Growth & Differentiation
1996
Expression
of 14-3-3y
and Cytokine-stimulated
Muscle Cells
Michael
Anne
V. Autieri,1
M. Romanic,
in Injured
Human
Dale S. Haines,
and Eliot H. Ohlstein
Pharmaceuticals,
King of Prussia,
Pennsylvania
19406
E.H.O.]
Abstract
One of the most critical
cellular events in disease
states such as vascular restenosis
is the cytokineinduced activation
of vascular smooth muscle cells
(VSMCs)
resulting
the molecular
in intimal thickening.
mechanisms
Identification
Received
5/13/96;
of VSMC
the initiation
8/26/96;
accepted
9/10/96.
of this article were defrayed
in part by the
This article must therefore
be hereby marked
in accordance
with 18 U.S.C. Section 1734 solely to mdi-
Biology,
actively
with
involving
balloon
several
cellular
events
in the restenotic
process
(4-6).
The identification
and characterization
of proteins involved
in the transduction
of proliferative
signals from the cell membrane to the nucleus represent an important
step in understanding
diseases in which cellular proliferation
is often the
principle cause of the pathology.
Numerous
ligands and their
corresponding
receptors
may be diverse, and various cornbinations
of signal
transduction
mechanisms
may
be
in-
volved in initiating cell proliferation.
However,
these signals
often converge into a few critical pathways
because all stimuli must eventually
mediate a commitment
to replicate DNA
and proceed
through
mitosis and division.
Growth factor
binding to their membrane
receptors activates a cascade of
cytoplasmic
kinases that are members of the Raf and MAPK
families. Activation
of MAPK by Raf is an essential link in the
transduction
of receptor-mediated
growth
factor-induced
cellular proliferation
(7-9).
The 14-3-3
protein family consists
of seven isoforms
of
highly conserved
acidic cytosolic
proteins (10-12). The 143-3 proteins
have diverse biochemical
functions
including
activation
of tyrosine and tryptophan
hydroxylases
(the ratelimiting enzymes
involved
in catecholamine
and serotonin
synthesis
in neurons and other endocrine
cells) and have
been implicated
in the inhibition
of protein kinase C. The y
isoform,
originally
thought
to be brain-specific,
has been
cloned
and shown
rat tissues
(13).
to be expressed
More
recently,
at very
it has been
low levels
shown
that
in some
mem-
bars of the 14-3-3
protein family associate
with the polyomavirus
middle T antigen, which is thought
to transform
cells by binding to proteins involved in cellular proliferation,
such as the sit family of protein tyrosine kinases (14). 14-3-3
protein homologues
have been identified
as required
for DNA
replication
in fission yeast, (1 4, 15) and in a yeast two-hybrid
2
The abbreviations
smooth
To whom requests
Molecular
associated
process
revised
for reprints should be addressed,
at Department
of
Research
Institute, 20 Pine Mill Road, Browns
Mills, NJ 08015. Phone: (609) 893-1016; Fax: (609) 893-2441.
1
formation
is a complex
Factor-
different cell types that secrete many different cytokines
and
growth factors seminal to the local inflammatory
response.
These factors include but are not limited to IL-i p,2 PDGF,
fibroblast
growth factor, and a number of colony-stimulating
factors and IFNs (1-3). The major cellular component
of the
atherosclerotic
lesion is the VSMC, which, in response
to
vessel wall trauma, migrates into the intimal layer and proliferates. It has been suggested
that cytokine-induced
activation and proliferation
of VSMCs in the media are the most
The costs of publication
payment
of page charges.
advertisement
cate this fact.
neointima
angioplasty
critical
of
activation
is
of vascular
restenosis.
In this report, we show that one 14-3-3
protein family isoform,
y, is transcriptionally
upregulated
in rat carotid arteries after balloon
angioplasty.
14-3-3y
protein induction
localizes to both
the media and neointima
in such injured vessels.
Because
it has been shown that some members of the
14-3-3 family
may play an important
role in cellular
proliferation
by binding to and activating
the protein
kinase Raf-1 and VSMCs constitute
the major cellular
component
of the restenotic
lesion, we investigated
the expression
of this message
in serum- and
cytokine-stimulated
human VSMCs.
Both serum and
selected
cytokines
induce 14-3-37
mRNA and protein,
the magnitude
of which correlates
with the degree of
cellular stimulation.
14-3-3i
mRNA, however,
does not
increase
when other cell types are stimulated
with
specific growth factors. Human tissue distribution
of
14-3-3’y
mRNA indicates that in contrast to other 14-33 proteins, the ‘y isoform is highly expressed
in VSMCs
and skeletal and heart muscle, suggesting
an
important
role for the y isoform in muscle tissue as
well. These results indicate that 14-3-3
expression
increases
in response
to vessel damage and
proliferative
signals and may implicate
a role for the ‘y
isoform
of 14-3-3
in VSMC activation
and metabolism.
crucial in understanding
and Growth
Smooth
Introduction
The resultant
Department of Molecular Biology, Deborah Research Institute, Browns
Mills, New Jersey 08015 [M. V. A.]; Department of Neoplastic Diseases,
Hahnemann
University,
Philadelphia, Pennsylvania 19102 [D. S. H.];
and Department
of Cardiovascular Pharmacology, SmithKuine Beecham
[A.M.R.,
Arteries
Vascular
Deborah
muscle
gen-activated
aldehyde-3-phosphate
hexamide;
used are: IL-i3,
interleukin
1(3; VSMC,
vascular
PDGF, platelet-derived
growth factor MAPK, mitoprotein kinase; UTR, untranslated
region; GAPDH,
glycercell;
PCNA,
dehydrogenase;
proliferating
cell nuclear
IL-2, interleukin
antigen.
2; CHX, cyclo-
1453
1454
in Restenosis
Induction
of i4-3-3y
system,
the 3 and
(in
and VSMCs
isoforms
have
been
activate
the protein
kinase
link in the ras signal transduction
gral
3-3 family
proteins
the protein
ture
have also been
kinases
for two
a putative
c-Bcr
i 4-3-3
kinase
isoforms
interact
(24).
with
purified
MAPK
has been
with
signaling
purified
kinase
3-3 proteins
characteristics,
proteins
found
(25), further
in regulation
solved
and
however,
has
differential
display
revealed
by phosphoserine
in concert
to activate
a cytosolic
a role for
proliferation.
been
G3PDH
ki-
None
reported
technology,
B
i4-
for the
in rat carotid
to both the
tissue.
Because
enotic
lesion
are VSMC
gated the role
human VSMCs
that
duced
in stimulated
cells.
i4-3-3y
found
also
mRNA
found
factors,
and
that
amounts
an additional
in heart
we further
are
of stimulation
skeletal
y isoform
muscle,
in muscle
in-
sug-
display
clone
was
originally
as an up-regulated
arteries
subjected
and
characterize
naive
rat carotid
three
time
identified
mRNA
cells.
to balloon
angioplasty
quantitate
this increase,
arteries
points
after
and from
balloon
carotid
arteries
angioplasty
rotid
To further
RNA from
analysis
(Fig. 1A).
blot normalized
Scanning
isolated
at
was size-fraction-
densitometric
to GAPDH
sion of i4-3-3y
increase
in day
content
in naive
1 vessels,
change
in basal
It was
arteries
levels
i4-3-3y
the
and
by
of i 4-3-3y
after
balloon
angioplasty
antibody.
Northern
analysis,
Similar
a low
protein
was present
in undamaged
majority
of which
localized
to smooth
medial
marked
medial
layer
(Fig.
induction
layer,
2A). Twenty-four
of i4-3-3y
continuing
through
h after
protein
day
was
basal
of
no
observed
3 (Fig.
rat
but
but
of protein
were
by cells
of the
to a lesser
degree
by both
and the cells
tissue-enhanced
RNA
with
than
that
injury. These data indicate
in balloon
angioplasty-damaged
layer
containing
from
the smooth
i4-3-3y
rat Ca-
muscle
cells
the
expression
of 1 4-3-3y,
3’ UTR
human
probe.
14and
observed
that
comprising
i 6 different
the i4-3-3y
that
(1 3), the greatest
in heart
cialized
liferating
and
amounts
skeletal
role for i4-3-3y
human
muscle
neointima.
tissues
filwere
Fig. 3 shows
bution
acid
to mRNA
of
a
in the
C,
of the 3 isoform
the
y isoform,
of the y isoform
muscle,
suggesting
that
sequence
is also
is conserved,
lesions
restenotic
lesion
and
are
tigated
the
human
VSMCs
with
grown
to
under
many
VSMC
role of i4-3-3y
just
a variety
of the
growth
regulatory
mecha-
expression.
primary
cell
cytokines
factors,
type
found
we
in vascular
injury
of growth
factors.
confluence,
but the
in Fig. 2 and differs
complex
nisms controlling
14-3-3
protein
family
Because
activated
VSMCs
are the
restenotic
nonproindicate
is not, and infer a funcThe human
tissue distri-
depicted
suggesting
were
a spe-
in terminally
differentiated
tissue as well. These data
this 3’ UTR nucleic
from
immu-
2, B and
layer
expression
pattern
of this transcript
tion for i4-3-3y
in muscle
tissue.
protein
injury,
amounts
medial
and expressed
in rat tissues
carotid
arteries,
muscle
of the
balloon
2E),
To determine
expressed
5-fold
above
level
in the
as in the rat, i4-3-3y
mRNA is also expressed
to a large
degree in human brain. However, in contrast to that observed
of this
Fig. 2 shows
(Fig.
screened
expres-
analysis
showed
only
after balloon
is induced
arteries
ters
iB).
vessels.
i4-3-3-y-specific
determined
Northern
vessels
progression
balloon-injured
before
with
(Fig.
to localize
in
a basal
vessels,
an approximately
and a decline
to slightly
expression
important
accumulation
analysis
indicated
basal levels in day 3 and day 7 vessels.
i4-3-3y
mRNA
from
sham
control
nostained
By day 7, appreciable
not
of the medial
ated and hybridized
with a radiolabeled
PCR product
representing
814 bp of the 3’ UTR of this gene by Northern
carotid
respectively).
7 days
protein
in rat carotid
(26).
total
and
developing
neointima
(Fig. 2D). At 2 weeks
postinjury,
3-3-)’ protein
was still detectable
in the medial
layer
by differential
transcript
of RNA from rat left common
carotid arteries
probe before ballon angioplasty
(Lane 1) and
angioplasty
(Lanes 2, 3, and 4, respectively).
carotid
arteries
was separated
on a 1.2%
was
rat
common
carotid arteries probed with i 4-3-3y
and )3-actin probes. Total
RNA (1 0 .Lg) from rat carotid arteries was separated
on a i .5% agarose/
formaldehyde
gel, transferred
to nitrocellulose,
hybridized,
and washed as
described
in “Materials
and Methods.”
neointima
i4-3-3y
#{149}
.
Fig. 1 . A, Northern analysis
probed with a i 4-3-3y
DNA
days 1, 3, and 7 after balloon
Total RNA (i 0 jig) from rat
expressed
Results
The
r
of this
of these
mRNA is
i4-3-3-y
4
agarose/formaldehyde
gel, transferred to nitrocellulose, hybridized,
washed
as described in “Materials
and Methods.”
A GAPDH
probe
used as a loading control. B, Northem
analysis
of sham-operated
rest-
investi-
expression
tissue,
and
role for the
in the
and that the extent
the degree
in human
3
i
?
‘ aCtin
previously
injury by stimulating
factors.
We demon-
protein
VSMCs
with
show
in large
gesting
growth
human
correlates
We
cytokines
of i4-3-3y
in vascular
with a variety of growth
strate
expression
of the
2
‘y sub-
arteries
after balloon
angioplasty,
medial
layer and neointima
in this
many
1
14- 3- 3
of these
we have
iS#{149}.
i4-3-3
shown that the mRNA of one i4-3-3
isoform,
y, is increased
5-fold 3 days after rat carotid
artery balloon
angioplasty
(26).
In this report,
we show
that i4-3-3y
protein
is also upregulated
localizing
4
#{149}
, 1,
14-3-3?
type.
Using
3
struc-
proteins,
suggesting
of cell
2
1
site and a phosphoryla-
i 4-3-3
were
A
and
with
The crystal
motif for cyclin-dependent
been shown
that some
Some
Ras protein,
kinase
to bind
to associate
(2i).
C binding
tion site within
a consensus
nases (22, 23), and it has
recognition
reported
and Bcr-Abl
isoforms
protein
shown
activity
of c-raf,
an intepathway
(i6-20).
14-
vitro)
serum-starved
further
inves-
by stimulating
VSMCs
in
in the
were
to quies-
Cell Growth
.
-.‘...
;-.
-
1455
-
_,;<
‘‘
.
s..’,
.
‘
‘-
‘
7’’
I,
,,.,u_...,.
)‘f
& Differentiation
.
‘
.,,.,
‘..
.t,I,I
.
..
Immunohistochemical
by immunohistochemistry
days after balloon angioplasty.
Fig. 3. Northern
tissue distribution
i4-3-3f3
mRNA
.
-.--
-.
I
.--;,._-
;_
Fig. 2.
protein
w
,:.
-.--.
-
analysis of i4-3-3y
expression
in rat carotid arteries after balloon angioplasty.
Tissue sections
were analyzed
for i4-3-3y
as described
in “Materials
and Methods.”
i 4-3-3y
expression
was monitored
in rat carotid arteries over a period of i 4
A, day 0; B, day 1 ; C, day 3; D, day 7; E, day i4; F, day 14 (negative
control).
Original magnification,
xiOO.
analysis of human
of i4-3-3y
and
expression.
Polya-
denylated mRNA (2 ,.Lg) from pancreas (1), kidney (2), skeletal muscle
9 1011
12345678
12
13
14
15
16
(3), liver (4), lung (5), placenta
(6),
brain (7), heart (8), peripheral
blood
lymphocyte (9), colon (10), small intestine
(1 1), ovary (12), testes (13),
prostrate
(14), thymus
(15), and
spleen
(16) was hybridized
with
each respective
as reported
purchased
cence,
previously.
from
and
__________________________________________
The blot was
Clontech.
exposed
to iO%
FCS,
PDGF,
IL-i 13, and
angio-
tensin II. Early activation
of these cells was confirmed
and
quantitated
by c-fos expression
and continued
stimulation
by [3H]thymidine
incorporation
Fig. 4A-D shows that, similar
quiescent
human
mRNA.
pression
In stimulated
correlated
Very
strong
duction
of mRNA
stimulatory
stimuli
of mRNA
VSMCs
however,
14-3-37
degree
of cellular
as 10%
(Fig. 4A). Weaker
FCS
elicited
was
lated
with
It was
mRNA exstimulation.
a robust
but significant
signal
by iO% FCS,
and magnitude
the
PDGF,
lL-if3,
and angiotensin
of i4-3-3y
mRNA expression
degree
of cellular
activation
in-
obtained
a
probe.
The degree of induction
of 1 4-3-3y
at the transcriptional
level was quantitated
by scanning densitometry,
and Fig. 5 is
a graphic comparison
of i4-3-3-y
and c-fos expression
in-
important
in mRNA
to demonstrate
was
translated
that
into
mRNA
this
i4-3-3y
level
PDGF
stimulation.
of protein
observed
human
by Northern
VSMCs
stimulation
basal
a greater
PDGF-stimulated
and
a greater
levels
VSMCs
with
the
analysis
demonstrated
and
stimulation.
increase
Starved
consistent
in-
protein.
Fig.
3-fold
at 8 and 16 h after
results
indicate
VSMCs.
mRNA
occurs
of mRNA
increase
increase
after
in serum-
stimulation,
that active
and
8 h of
after
cells demonstrated
than
2-fold
increase
(Fig. 6B). These
i4-3-3y
a basal
levels
4). FCS-stimulated
a 2-fold
than
in serumafter serum
exhibited
basal
(Fig.
by
levels
inducible
6A shows
immunoblot
analysis
of i4-3-3y
starved VSMCs and VSMCs at various points
and
induction
with
crease
II. The
corre-
as measured
expression
and indicated
that i4-3-3y
increase
in response
to proliferative
signals.
Angiotensin
II, at 1O
M/mI, was not a potent stimulator
of
VSMCs and, of the four stimulatory
agents examined,
induced the weakest
induction
of both c-fos
and i4-3-3y
4D). No hybridization
duced
pattern
c-fos
not shown).
to heart and skeletal muscle,
express a basal level of 14-3-3y
VSMCs,
with the
such
(data
was elicited
by both PDGF and IL-i 3, both potent
agents for VSMCs (Fig. 4, B and C; Refs. i and 2).
mRNA (Fig.
i4-3-3p-specific
I
probe as described
in “Materials
and Methods.”
The respective
size of each transcript
(143-3.y,
34 kb; 14-3-3/3,
2.8 kb) was
1 6 h of
a 3-fold
above
respectively
translation
cytokine-activated
of
1456
Induction
of 14-3-3y
in Restenosis
0
and VSMCs
1.5
0.5
3.0
4.5
B
8.0
0.5
14.3.3.y
14.3.3.y
1.5
3.0
4.5
8.0
Northem analysis of huVSMCs stimulated
with
10%
FCS A), iO
u/mI PDGF
(B), io
u/mI IL-1f3 (C), or iO
WmI angiotensin II (D) hybridized
Fig. 4.
*‘
man
---
C-foe
C-los
with
.
G3PDH
-
G3POH
C
0.5
14.3.3.’)?
1.5
,.
3.0
4.5
D
8.0
0.5
14.3.3.y
-
w
w
w
1.5
w
w
3.0
4.5
8.0
stripped
bridized
-
and
c-fos
DNA
and
with
sequentially
hythe
respective
probes as described in “Materials and Methods.” Autographic
exposure time was 16 h for all
probes.
C-foe
C-fOS
14-3-3y
probes. A GAPDH probe was
used as a loading control. Numbers at the top of each lane indicated the time of stimulation
before RNA isolation. Total RNA
(25 p.g) was analyzed
in each
lane, and the same filter was
-
G3PDH
G3PDH
w
w
w
w
w
-
indicate that induction
of l4-3-3y
mRNA is not a general
response to cellular proliferation.
As with other cell types, serum or growth factor stimulation
of VSMCs
in culture
induces
cycle and transcription
cycle-dependent
>.
genes
through
the cell
of well-characterized
(30, 31). Immediate
early
by their
insensitivity
to the protein
inhibitor
whereas
transcription
of the intermediate
CHX,
then
stimulated
cell
genes
characterized
genes is inhibited.
affected
by CHX,
.D
a progression
of a number
early
To determine
if i4-3-3y
transcription
human VSMCs were serum-starved
with
1 0%
are
synthesis
FCS for 8 h in the presence
is
and
of 500
CHX. Fig. 8A shows that this concentration
of CHX had no
effect on basal levels of i 4-3-3y
but inhibited expression
at
8 h by approximately
60% (Fig. 8B), suggesting
multiple
CHX-sensitive
and -insensitive
factors are responsible
for
nM
2
i
3
4
i 4-3-3y
5
Scanning
densitometric
analysis
of Northern
blots of l4-3-3y
mRNA expression in stimulated VSMCs. Abscissa, numbers correspond
to c-los at 30 mm of stimulation and i4-3-3y
at 8 h of stimulation and are
normalized
for GAPDH expression.
Lane 1 , starved VSMCs; Lane 2, 10%
FCS; Lane 3, PDGF; Lane 4, IL-i f3; Lane 5, angiotensin
II.
Fig. 5.
Other
inducible
if induction
Mouse
of
T lymphocytes
not
to
VSMCs.
a good
model
ability
to enter
cell
cycle
IL-2 stimulation
(27-29).
L2 cells were stimulated
with
of rlL-2,
these
growth
induced
cells
and
a concentration
through
to quiescent
detectable
the cell
to study
the
of IL-2
cycle
was
(42). Fig.
cellular
used
factor-stimulated
VSMCs,
L2 cells
IL-2 addition.
Activation
of these
quantitated
by PCNA and p-actin
which increase in such stimulated
1 4-3-3y
either
to
7 indicates
message
injury of the arterial
thesis
of various
recruited
immune
escent
VSMCs
smooth
muscle
modulation
VSMCs,
a basal level of 1 4-3-3y
in unstimulated
L2 cells. However,
in lL-2-stimulated
Discussion
Mechanical
in localized
syn-
cytokines
by both the medial
VSMCs
cells (2, 4, 5). Activation
of normally
and
qui-
to these
cell
wall results
cytokines
response
is an example
to vascular
trauma.
of the
In this
study, we have shown the expression
of l4-3-3y
mRNA to
be up-regulated
in rat carotid arteries 1-3 days after balloon
angioplasty
(Fig. iA). This is the first reported
transcriptional
to determine
stimulated
of their
that, similar
mRNA
was
unlike
examined
provide
i 00 units
drive
were
is specific
because
proliferation
upon
cell systems
i4-3-3y
transcription.
is
8 or 24 h after
cells was confirmed
and
gene expression,
both of
cells (27-29). These data
of a 14-3-3
protein
in a disease
process.
Recent observations
show that the 1 4-3-3
and
isoforms have been identified as an integral link in the ras and
MAPK signal transduction
pathways
(1 4-25). None of these
observations
have been reported for the y subtype. In light of
these reports of 14-3-3 protein involvement
in cellular proliferation,
and given the fact that VSMC proliferation
is a
hallmark
of vascular
restenosis,
it may
prising
that i4-3-3y
Immunohistochemistry
mRNA is increased
of balloon-injured
a dramatic
of 1 4-3-37
induction
protein
not
be entirely
sur-
in this tissue.
arteries
revealed
in the medial
layer of
Cell Growth
1
A
2
& Differentiation
1457
3
-
2
ss
u5c2
Q
0
U..
LL:
14-3-3Y
(is
Q
0.
U
5.
97.4
-
-46
PCNA
I
-30
-
1 actin
14.3
-
B
28S
oE
.2
11
18S
Fig. 7. Expression
RNA was isolated
of
from
IL-2 (100 units/mI)
mRNA in IL-2-stimulated
L2 cells. Total
the murine T helper cell 12 at varying times after
i4-3-3’y
stimulation
and hybridized
with a 14-3-3y-specific
probe. RNA was isolated
from cells at the following
time points: 1 , quiescent; 2, 8 h; and 3, 24 h. Total RNA (25 g) was analyzed
in each lane,
and the same filter was stripped
and sequentially
hybridized
with the
respective
probes as described
in “Materials
and Methods.”
Probes for
Fig. 6.
Expression
of i4-3-3y
protein
in human
VSMCstreated
with FCS
protein
levels by Western analysis as described
in “Materials
and Methods.”
Lane
1 , control
cells cultured
for 16 h in serum-free
conditions;
Lane 2, cells
treated with 10% FCS for 8 h; Lane 3, cells treated with i09 M PDGF for
8 h; Lane 4, cells treated
with 10% FCS for 1 6 h; Lane 5, cells treated with
io-9 u PDGF for 16 h. Molecular mass markers (in kDa) are as indicated.
B, densitometric
analysis
of 14-3-3y
expression
detected
using the enhanced chemiluminescence
method. Values expressed
are the average of
two experiments
and are displayed as the relative level of expression
normalized
to the control.
and PDGF. A, cell extracts (7.5 d each) were analyzed for 1 4-3-3y
PCNA and 13-actin were used to show cellular
exposure
time
was
1 6 h for
PCNA
and
f3-actin
activation.
probes
Autographic
and
48 h for
probe.
i4-3-3’y
it was
shown
that the f3 and ‘q isoforms
oid in their expression,
thymus,
whereas
highest
expression
the
with the highest
y isoform
in the
brain
was
were primarily lymphlevels in spleen and
brain-specific,
and
significant
with
amounts
the
in
skeletal muscle (1 3). The distribution
of 14-3-3y
in human
tissue has not previously
been described.
Fig. 2 shows that
rat carotid
At
arteries
later
time
i day after balloon
points,
1 4-3-3y
present
in cells of the developing
virtually
every
cell
of the
angioplasty
immunoreactivity
neointima,
neointima
(Fig. iB).
in
was
levels in many tissues, with the greatest amounts
in heart,
skeletal muscle, and brain. This tissue distribution
is unique
among the 1 4-3-3 family isoforms and suggests
the presence of interesting
and complex
cis sequences
and trans
factors governing
14-3-3 family transcription.
Both heart and skeletal tissue are comprised
of terminally
differentiated
nonproliferating
muscle
cells, and at first
glance, the high levels of 14-3-3y
mRNA expression
in these
tissues are unexpected.
However, it has been recently demonstrated
that MAP kinase is activated
in and may play an
important
role in the regulation of muscle contraction
(34). In
VSMCs, a natural substrate
for MAP kinase is caldesmon,
and it has been shown that phosphorylation
of caldesmon
by
MAP kinase modulates
the effects of caldesmon
on actinmyosin interactions
(34-37). The constitutive
expression
of
also
and at day 7,
expressed
appreciable
amounts of 14-3-3y
protein. At 2 weeks after balloon angioplasty, i4-3-3y
levels seemed
to decline,
with not every cell
in the neointima
expressing
to note
majority
this
that
time
the
point
seemed
i4-3-3y
of cells
protein.
that
to localize
It is interesting
expressed
1 4-3-3y
to the luminal
side
at
of the
neointima,
an area where a greater proportion
of stimulated
cells are located (5, 6). This temporal rise and fall of 14-3-3y
protein in injured rat carotid arteries may reflect the activation
state of the cells of the neointimal
layer, coinciding
with
localized inflammatory
cytokine levels.
Levels of most 14-3-3
proteins
seem to be tissue-restricted
(1 0, 1 1 , 32, 33). Although
most isoforms
are expressed at low levels in many tissues, the levels of expression
differ
most
soluble
considerably.
isoforms
protein.
There
are
in brain
tissue
Indeed,
in rat multiple
very
at levels
large
amounts
as high
as
tissue
Northern
of
1%
of
blots,
human
i4-3-3y
tissue,
14-3-3y
in muscle
tissue,
mRNA
is expressed
serum-starved
at
VSMCs,
damaged arteries (Figs. 2A, 3, and 4A, respectively)
a hypothesis
of dual roles for i4-3-3-y
in VSMCs
that
proposed
for MAPK:
a fundamental
role
very
and
low
un-
supports
similar to
in maintaining
1458
Induction
of i4-3-3y
in Restenosis
and VSMCs
A
temporal
1234
1O%FCS
CHX
-
-
+
+
-
+
-
+
events
cycle.
The
that occur
temporal
characteristic
genes
of their
14-3-3y
synthesis.
expressed
A characteristic
by
#{149}I
#{149}#{149}
whereas
the
Low
B
40
doses
expression
30
used
of CHX
a
of
for
1
2
3
4
RNA
phenotype
during
role
coding
function
response
as tranin the cell
of protein
genes
synthesis
is their
inhibitor
early
normal
in cellular
a good
CHX,
genes
is often
was
54%.
to study
cell cycle
upon
mitogen
respond
to growth
factor
of the
cellular
to dedifferentiate
used
(38) and,
to dissect
at this
con-
kinase
(39).
as high as i g/ml
synthesis
(30).
In rat
has been
In our
hands,
i j.tg/ml showed
toxic effects
presence
of 500 ng/ml of CHX,
maintained,
The
been
above
In the
fact
and
that
i4-3-3’y
expression
expression
was
not
was
on
cell
inhib-
unaffected
or
Although
this does not rule out the possibility
factor stimulation
leads to increased
stability
of
the i 4-3-3y
message,
it does
novo synthesized
transcription
tion.
Conversely,
in
there
is a 2.5-fold
increase
serum-induced
vessel
the
stimulation.
VSMCs
are able
signals
released
in response
serum-stimulated
above
to
to
indicate
i4-3-3y
CHX-treated
baseline
levels,
is not totally
cells,
indicating
dependent
synthesis.
It is also feasible
that
baseline
synthesis
or an additional
factors
are posttranscriptionally
tion to increase
transcription.
prolifera-
and reenter
indicate
the necessity
of de
factors
for optimal
transcrip-
transcription
protein
responsible
for
activation.
model
ability
function
have
thymidine
of protein
de novo
of their
protein
G1 progression
inhibition
stimulation.
that growth
that
provide
often
a
early
superinduction
was not observed
indicates
that i4-3-3y
is
not an immediate
early gene in growth
factor-induced
VSMC
Fig. 8. A, effect of CHX on accumulation
of i4-3-3y
mRNA.
Human
VSMCs were serum-starved
for 48 h and then stimulated
with i 0% FCS
in the presence
or absence
of 500 ng/ml CHX for 8 h, and RNA was
analyzed
by Northern
analysis.
A GAPDH
probe was used as a loading
control.
B, scanning
densitometric
analysis of Northern
blots of i4-3-3y
mRNA expression
in serum-stimulated
CHX-treated
human VSMCs. Abscissa,
numbers
correspond
to i4-3-3y
at 8 h of stimulation and are
normalized
for GAPDH expression.
because
immediate
inhibits
protein
synthesis
by 50% within 1 h
prevents
cell cycle
progression
as well as
VSMCs.3
viability
1
ited
VSMCs
and
CHX at concentrations
human
tion
The
are induced
later
is independent
(iOO ng/ml)
CHX concentrations
20
an additional
is often
of immediate
factor-induced
VSMCs,
C
VSMC
of the cell
genes
of early
the
expression
centration,
CHX
and completely
and
phases
inhibited.
growth
the
the early
of these
function.
for genes that
their transcription
superinducibility
G3PDH
protein
are transiently
scription
factors
cycle. As such,
during
expression
on
factors
set of
modified
by serum stimulaTaken together,
these results
that more than one factor is involved
expression,
at least one of which
in regulation
is CHX-insensi-
of
tive.
The pleiotropic
functions
transduction
of proliferative
of i4-3-3
family
proteins
in
signals
are just beginning
to be
cells, the pattern
and magnitude
of 1 4-3-3y
mRNA expression correlate
with the degree of cellular stimulation
as meas-
investigated.
Although
the
have not been investigated,
specific
functions
of
it is likely that i 4-3-3y
ured
role in both
injury. The local accumulation
injury has profound
effects
by c-fos
expression
sion (Fig.
with
the
degree
basal,
indicate
mRNA
is
suggesting
cytokine-driven
report of i4-3-3y
protein,
in vascular
In cultured
that
of these
of i4-3-3y
i4-3-3y
cytokines
with inducible
of this expression
of stimulation
analysis
of
above
and
is associated
4). The extent
munoblot
lation
expression
of these
on VSMCs.
i4-3-3y
mRNA
cell cycle
correlates
progresclosely
cells.
at
a specialized
for
human
VSMC
activation.
This
mRNA expression
associated
mal cellular
proliferative
state. Although
i4-3-3y
in VSMCs
by growth
factors,
it is not inducible
cytes
by the primary
T-cell
the precise
in stimulated
molecular
VSMCs
rant
investigation.
further
growth
mechanisms
and skeletal
factor
of expression
and cardiac
of a number
of genes
has
been
used
normal
and aberrant
Raf activation
occurs
after the addition
mm postinduction
levels
levels
reason
for this
though
it is possible
i4-3-37
in
is the first
with a noris induced
in T lymphoof i4-3-3y
muscle
war-
occur
after
this
and
the
the
within
time,
increase
that
Raf activation
protein
Raf. Therefore,
factor-driven
other than
and growth
stimulated
i4-3-3
VSMCs
isoforms
cidation
of the
cular restenosis,
not
but
3
Unpublished
only
underscore
infer
the
sole
to Raf activity.
that
Al-
in stimu-
is that increased
y isoform
binds
a
the muscle
tissue
speciexpression
of 1 4-3-3y
in
functional
the
differences
importance
role of i4-3-37
in muscle
and VSMC
pathophysiology.
observations.
at 5 mm
decreased
10
i4-3-3y
protein
is delayed
possibility
that the
a
In HeLa
peaks
unlikely
is to contribute
more likely
indicates
ficity
3 mm and
it seems
i4-3-3y
plays
proliferation.
is significantly
increased
lated VSMCs,
the
i4-3-3y
expression
a progresto map
VSMC
of serum and
(40). Because
trans-
IL-2 (Fig. 7). Clearly,
Serum stimulation
of VSMCs in culture induces
sion through
the cell cycle
and cell proliferation,
induction
that
significant
role
cells,
Fig. 6, an im-
indicates
occurring
human
in
of the elu-
metabolism,
vas-
Cell Growth & Differentiation
Materials
and Methods
Rat Left Common
Carotid
Artery
carotid
artery balloon angioplasty
Balloon
Angloplasty.
Left common
was performed under aseptic conditions on male Sprague-Dawley
rats (350 g; Charles River Breeding Laboratories, Wilmington, MA) under sodium pentobarbital
anesthesia
(65
mg/kg, i.p.; Steris Laboratories, Phoenix, AZ) as described
previously (26,
41). Briefly, the left extemal carotid artery was identified
and cleared of
adherent tissue, allowing the insertion of a 2-F Fogarty arterial embolectomy catheter(model
12-060-2F; Baxter Healthcare, Santa Ana, CA). The
catheter was guided a fixed distance down the common carotid artery to
the aortic arch, inflated with a fixed volume of fluid, and withdrawn to the
site of insertion.
This procedure was performed
a total of three times.
Once completed, the catheter was removed, and the wound was closed
(9-mm autoclips; Clay Adams, Parsippany, NJ) and swabbed with Povadyne surgical scrub (7.5% povidone-iodine;
Chaston, Dayville, CT). Animals were housed in Plexiglas cages under a 12-h light/dark cycle with
access to standard laboratory chow and drinking water ad ilbitum until
required
for tissue collection.
To isolate the carotid arteries, rats were exsanguinated
via the vena
cava under barbiturate anesthesia (100 mg/kg, i.p.). Left common carotid
arteries
were rapidly cleared of adherent tissue in situ, isolated, and
placed directly in guanidine thiocyanate (Promega, Madison, WI). These
vessels were then immediately frozen in liquid N2 and stored at -80#{176}C
until required for RNA isolation. For subsequent Northern analysis, tissues
were examined
from
undergone
angioplasty
the differential
fashion.
naive animals
6 h, 3 days,
expression
Northern
analysis
(control)
and from animals
that had
and 14 days before in order to study
of transcripts
was also
in a more detailed
performed
on sham
vessels.
temporal
All sur-
gical procedures were performed in accordance with the guidelines of the
Animal Care and Use Committee
of SmithKline Beecham Pharmaceuticals
and the American Association
for Laboratory Animal Care.
lmmunohistochemlstry.
Left common
carotid
arteries that had undergone balloon angioplasty were removed from rats afterin situ perfusion
fixation
(90 mm Hg) using
10%
(w/v) phosphate-buffered
formalin
(Baxter
Scientific). After standard histological processing and embedding in paraffin, 6-tm-thick
sections were prepared for immunoperoxidase
staining
using the Vectastain
Elite ABC kit (Vector Laboratories)
according
to the
manufacturer’s
instructions.
Briefly, endogenous
peroxidase
was
quenched
with 0.3% H202 in methanol
for 30 mm. Nonspecific
immunoglobulin
binding sites were blocked
with normal goat serum for 1 h, and
then the sections were incubated with the primary antibody [rabbit anti14-3-3y (C-16), 0.1 g/ml; Santa Cruz, Inc.] for 1 h at room temperature.
This antibody
is raised against
amino acids mapping
at the carboxyl
terminus
of 14-3-3’y and is partially reactive to 14-3-3t but nonreactive
with most 14-3-3
protein members.
As a negative control, serial sections
were incubated
the primary
biotinylated
oratories)
with rabbit IgG (0.1 &g/ml; Vector Laboratories)
antibody.
The sections
goat anti-rabbit
followed
were then
IgG secondary
by 30 mm of incubation
reagent solution. Immunoglobulin
bation with 3,3’-diaminobenzadine
incubated
antibody
(Vector Laboratories).
hematoxylin,
cleared,
upon incuSections
were
culture were provided by Clonetics Corp. (San Diego, CA)and subcultured
in growth medium as described
previously (42, 43). These cells test
for smooth
muscle
a-actin
and negative
for factor
VIII. The growth
media were changed
every other day until just under confluence. Cells
used in the described
studies.
Preconfluent
VSMCs wereserum-starved
for48 h in DMEM, then exposed to 10% FCS,
io- u/mI PDGF, i0
u/mI IL-1f3, or io
u/mI angiotensin II for the
times indicated.
PDGF and lL-1 were purchased from Life Technologies,
Inc. (Bethesda, MD), and angiotensin II was purchased
from ICN Biochemicals (Irvine, CA). The derivation and maintenance of L2 cells, an
from
passages
5-9
were
alloreactive
mouse
helper T-ceII clone of B6 origin (H-2”),
have been
described
elsewhere (27, 28). Briefly, cells were grown in DMEM (Bioproducts,
Walkersville,
MD) supplemented
with 100 units/mI
penicillin,
100 mg/mI streptomycin,
4 mu glutammne,
50 mu 2-mercaptoethanol,
10% heat-inactivated
FCS (Hyclone Laboratories, Logan, UT) - DMEM/
CM. For expansion, 12 cells at a concentration
of 1 x 1 06 cells/mI were
cocultured
with
irradiated
allogeneic
CBA/J
spleen
PCNA.
RNA Isolation
and Northern
Analysis.
For each time point studied,
four or five left carotid arteries were pooled, or VSMCs from culture were
isolated, and total RNA was obtained according to standard
methods as
described
previously
(26). Equal amounts of RNA were loaded and separated on a 1 .3% agarose/formaldehyde
gel, transferred to nitrocellulose,
and hybridized
(0.25 u NaCI, 1 % SDS, 50% formamide,
2 x Denhardt’s
solution, 25 L9 denatured
salmon sperm DNA, and 5% dextran sulfate at
42#{176}C
ovemight) with the indicated probe. Blots were washed under high
stringency (0.2 x sodium citrate and 0.1 % SDS, 65#{176}C)
and exposed to
film for 6-48 h at -80#{176}C.All probes were [a32P]-labeled
by the random
priming method (Bcehringer
Mannheim,
Indianapolis,
IN; all Isotopes were
from Arnersham,
Arlington
Heights,
IL). The same filter was stripped
and
subsequently hybridized with the various DNA probes. The l4-3-3y probe
consisted of 814 bp of 3’ UTR and was obtained from differential display
described
previously
(26), and the human c-fos probe was a gift of Dr. D.
Haines (Hahnemann University, Philadelphia, PA). The PCNA and -actin
probes have been described
(29), and the GAPDH probe was generated
from PCR amplimers (Clontech, Palo Alto, CA). Relative intensities of
hybridization
signals
were
Scan software;
Scanalytics,
obtained
by densitometric
scanning
(RFLP-
Inc.) of autoradiograms
exposed within the
linear range of the film (Kodak X-OMAT). Human multiple-tissue
Northern
blots were purchased from Clontech.
Western
Analysis.
To prepare cell extracts, human VSMCs were cultured and treated as described above, washed with PBS, and treated with
1 ml of ice-cold lysis buffer (1% NP4O, 0.1 % SDS, and 0.01 % sodium
azide in PBS). Cells were scraped from the flasks and lysed further by
withdrawing
the
cells
through
a 21 -gauge
needle
three
times.
Lysates
were incubated
on ice for 30 mm, then 1 mu phenylmethylsulfonyl
fluoride
was added, and the samples were centrifuged at 14,000 rpm for 1 0 mm
at 4#{176}C
and stored until use at -20#{176}C.
Equal volumes (7.5 s.d)of cell extracts were electrophoresed
through a
12% polyacrylamide gel under reducing conditions (44) and transferred to
a nitrocellulose membrane. Membranes were blocked overnight at 4#{176}C
with 5% nonfat powdered milk in TBST buffer [0.1 u Tris-HCI (pH 8.0)
containing 1.5 u NaCI and 0.5% Triton X-100). Primary antibody
to 143-3’ (described above) diluted to 1 pg/mI in TBST was incubated
for 1 h
at 25#{176}C,
and the membrane was washed three times with TBST and
incubated for 30 mm with secondary antibody
(donkey anti-rabbit IgG
conjugated
to horseradish
peroxidase; Amersham) diluted 1 :3000 in
TBST. The membrane was washed as above and developed
using the
enhanced chemiluminescence
method (Amersham) according to the
manufacturer’s instructions. The levels of intensity of each band relative to
the control were determined and quantitated with a Molecular Dynamics
densitometer (Molecular Dynamics, Sunnyvale, CA).
Elite ABC
were visualized
washed,
counterstained
with Gill’s
mounted
with
Aquamount
(Polysciences),
and examined by light microscopy.
Cell Cufture.
VSMCs from human aorta as cryopreserved secondary
positive
centration
of 4 x 1 o cells/mI in 1 0 ml DMEM/CM
containing
1 00 Cetus
units/mI of highly purified recombinant
human IL-2 (Cetus). Proliferation
of
these cells was verified by thymidine incorporation and the expression of
(1:200; Vector Lab-
with the Vectastain
complexes
instead of
for 30 mm with a
cells
(H-2’)
at a con-
1459
References
1 . Ross, R. The pathogenesis of atherosclerosis:
19905. Nature (Lond.), 362: 801-809, 1993.
2. Nilsson,
Cardiovasc.
J. Cytokines
and smooth muscle
189, 1992.
a prospective
for the
cells in atherosclerosis.
Res., 27: 1184-1
3. Clowes A. W., Reidy, M. A., and Clowes, M. M. Mechanisms
after arterial injury. Lab. Invest., 49: 208-215,
1983.
of stenosis
4. Uu, M. W., Roubin, G. S., and King, S. B. Restenosis after coronary
angioplasty:
potential biologic determinants and role of intimal hyperplasia. Circulation, 79: 1374-1378, 1989.
5. Austin, G. E, Ratliff, N. B., Hollman, J., Tabei, S., and Phillips, D. F.
Intimal proliferation of smooth muscle cells as an explanation
for recurrent
coronary artery stenosis after percutaneous
transluminal
coronary angioplasty. J. Am. CoIl. Cardiol., 6: 369-375, 1985.
6. O’Brien, E. R., Alpers, C. E., Stewart, D. K., Ferguson, M., Tran, N.,
Gordon, D., Benditt, E. P., Hlnohara, T., Simpson, J. B., and Schwartz, S.
M. Proliferation in primary and restenotic
coronary
atherectomy tissue.
Circ. Res., 73: 223-231 , 1993.
7. Feig, L A. The many roadsthatlead
767-768,
1993.
to Ras. Science (Washington),
260:
14
Induction
of 14-3-3-y in Restenosis
and VSMCs
8. Marx, J. Forging a path to the nucleus.
1588-1590,
Science
(Washington),
260:
1993.
9. Roberts, T. M. A signal chain of events. Nature (Lond.), 360: 534-535,
1992.
10. Aitken,
Rosenboom,
family
1992.
A., Collinge,
P. H. 14-3-3
of eukaryotic
D. B., van Heusden,
B., Isobe,
T., and
proteins:
a highly conserved,
widespread
proteins. Trends Biochem. Sci., 1 7: 498-501,
11. Aitken, A., Arness, B., Howell, S., Jones, 0., Martin, H., Patel, V.,
Robinson, K., and Toker, A. The role of specific isoforms of 14-3-3 protein
in regulating protein kinase activity in the brain. Biochem. Soc. Trans., 20:
607-611, 1992.
12. Isobe, T., Ichimura, T., Sunaya, T., Okuyama, T., Takashi, N., Kuwano,
R., and Takahashi, V. Distinct forms of the protein kinase-dependent
activator
of tyrosine
and tryptophan
hydroxylases.
J. Mol. Biol., 217:
125-132,
1991.
rnRNAs induced by rat carotid artery balloon angioplasty.
656-671, 1995.
Lab. Invest., 72:
27. Sabath, D. E., Podolin, P. L, Comber, P. G., and Prystowsky, M. B.
cDNA cloning and characterization
of interieukin 2-induced genes in a
cloned T helper lymphocyte. J. Biol. Chem., 265: 12671-12678,
1990.
28. Glasbrook, A. L, Sarmiento, M., Loken, M. R., Dialynas, D. P.,
Quintans, J., Eisenberg, L, Lutz, C. T., Wilde, D., and Fitch, F. W. Murine
T-lymphocyte
clones with distinct immunologic functions. Immunol. Rev.,
54: 225-266,
1981.
29. Shipman, P. M., Sabath, D. E., Fischer, A. H., Camber, P. G., Sullivan,
K., Tan, E. M., and Prystowsky, M. B. Cyclin mRNAand protein expression
in recombinant interleukin 2-stimulated cloned murine T lymphocytes.
J.
Cell. Biochem.,
38: 39-48, 1988.
30. Gadeau, A. P., Campan, M., and Desgranges, C. Induction of cell
cycle-dependent
genes during cell cycle progression of arterial smooth
muscle cells in culture. J. Cell. Physiol., 146: 356-361 , 1991.
13. Watanabe, M., Isobe, T., Ichimura, T., Kuwano, R., Takashi, V., and
Kondo, H. Molecular cloning of rat cDNAs for f3 and y subtypes of 14-3-3
protein and developmental changes in expression of their mRNAs in the
nervous system. Mol. Brain Res., 17: 136-146,
1993.
31 . Campan, M., Desgranges, C., Gadeau, A. P., Millet, D., and Belloc, F.
Cell cycle-dependent
gene expression in quiescent
stimulated
and asynchronously cycling arterial smooth muscle cells in culture. J. Cell. Physiol.,
150: 493-500,
1991.
14. PaIlas, D. C., Fu, H., Haehnel, L C., Weller, W., Collier, R. J., and
Roberts, T. M. Association of polyomavirus
middle tumor antigen with
32. Ceiis, J. E., Dejgaard, K, Madsen, P., Leffers, H., Gesser, B., Honors,
B., Rasmussen, H. H., Olsen, E, Lauridsen, J. B., and Ratz, G. The MRC-5
human embryonal lung fibroblast two-dimensional
gel cellular protein
database: quantitative identification of polypeptides whose relative abundance differs
between
quiescent, proliferating,
and SV4O-transformed
cells. Electrophoresis,
1 1: 1072-1 113, 1990.
14-3-3
proteins.
Science
(Washington),
265:
535-537,
1994.
15. Ford, J. C., Al-Khodairy,
F., Fotou, E., Sheldrick, K. S., Griffiths, D.
J. F., and Can’, A. M. 14-3-3 protein homologs required for the DNA
damage checkpoint
in fission yeast. Science (Washington),
265: 533534, 1994.
16. Morrison, D. 14-3-3: modulators
of signalling proteins? Science
(Washington),
266:
56-57,
1994.
17. Freed, E., Symons, M., Macdonald,
S. G., McCormick,
F., and
Ruggieri, A. Binding of 14-3-3 proteins to the protein kinase Raf and
effects on its activation. Science (Washington), 265: 1713-1716,
1994.
18. Ire, K, Gotoh, V., Yashar, B. M., Errede, B., Nishida, E., and
Matsumoto, K. Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase. Science (Washington), 265: 1716-1719,
1994.
19. Fu, H., Xia, K., Pallas, D. C., Cui, M. C., Conroy, K., Narsimhan, R. P.,
Mamon, H., Collier, R. J., and Roberts, T. M. Interaction of the protein
kinase Raf-1 with 14-3-3
proteins.
Science (Washington),
266: 126-129,
1994.
20. Fantl, W. J., Muslin, A. J., Kikuchi, A., Martin, J. A., MacMicol, A. M.,
Gross, R. W., and Williams, L T. Activation of Raf-1 by 14-3-3 proteins.
Nature (Lond.), 371: 612-614, 1994.
21 . Reuther, G. W., Fu, H., Cripe, L D., Collier, J. R., and Pendergast, A.
M. Association of the protein kinases c-Bcr and Bcr-Abl with proteins of
the 14-3-3 family. Science (Washington),
266: 129-143, 1994.
22. Xiao, B., Smerdon, S. J., Jones, D. H., Dodson, G. G., Soneji, V.,
Aitken, A., and Gamblin, S. Structure of a 14-3-3 protein and implications
for coordination
of multiple signalling pathways. Nature (Lond.), 370:
188-191,
1995.
33. Rosenfeld,
tribution,
J., Petosa, C., Collier, R. J., Fu, H., and
structure
of the
isoform of the 14-3-3 protein.
Uddington,
R. Crystal
Nature (Lond.), 376: 191-194,
1995.
24. Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. Interaction
of 14-3-3 with signaling proteins is mediated by the recognition
of phosphoserine.
Cell, 84: 889-897,
1996.
B., and Loose-Mitchell,
of rat 14-3-3
protein.
FASEB
D. Cloning, disJ., 5: A834, 1991.
34. Katoch,
S. S., and Moreland,
R. S. Agonist
and membrane
depolarization-induced
activation of MAP kinase in the swine carotid artery.
Am. J. Physiol.,
269: 222-229,
1995.
35. Adam,
sequences
L P., Gapinski,
In h-caldesmon
C. J., and Hathaway,
D. R. Phosphorylation
from phorbol
ester-stimulated
canine aortas.
1992.
FEBS Left., 302: 223-226,
36. Adam, L P., Haebe,le, J. R., and Hathaway, D. R. Phosphorylation
of
caldesmon in arterial smooth muscle. J. Biol. Chem., 264: 7698-7703,
1989.
37. Adam, L P., and Hathaway, D. R. Identification of mitogen-activated
protein kinase phosphorylation
sequences
in mammalian h-caldesmon.
FEBS Left., 322: 56-60,
1993.
38. Pardee,
proliferation.
A. B. A restriction point for control of normal animal cell
Proc. NatI. Acad. Sci. USA, 71: 4901-4905,
1974.
39. Coppock,
during
40.
D. L, and Pardee, A. B. Control of thymidine
the cell cycle.
Hipskind,
Mol.
Cell. Biol.,
R. A., Baccarmni,
7: 2925-2932,
M., and Nordheim,
kinase
mRNA
1987.
A. Transient
activation
of RAF-i , MEK, and ERK2 coincides kinetically
with temary
complex
factor phosphorylation
and immediate early gene promoter activity. Mol.
Cell. Biol., 14: 6219-6231 , 1994.
41
23. Uu, D., Bienkowska,
G. C., Samborn,
and expression
.
Ohlsteln,
E. H., Douglas,
S. A., Sung,
C. P., Vue,
T. L,
Louden,
C.,
Meth, A., Poste, G., Ruffolo, R. R., Jr., and Feuerstein, G. 1 Carvedilol,
a cardiovascular drug, prevents vascular smooth muscle cell proliferation,
migration,
and neointima
formation
Aced. Sci. USA, 90: 6189-6193,
following
1993.
vascular
injury.
Proc.
NatI.
42. Vue, T. L, McKenna, P. J., Gu, J. L, and Feuerstein, G. 1 Interleukin-8 is chemotactic for vascular smooth muscle cells. Eur. J. Pharmacol.,
81-84,
1993.
25. Shimizu, K., Kuroda, S., Vamamori, B., Matsuda, S., Kaibuchi, K.,
Vamauchi, T., Isobe, T., Irie, K., Matsumoto, K., and Taka, V. Synergistic
activation by tas and 14-3-3 protein of a mitogen-activated
protein kinase
kinase kinase named Ras-dependent
extracellular signal-regulated
kinase
kinase stimulator.
J. Biol. Chem., 269: 22917-22920,
1994.
240:
26. Autieri, M. V., Feuerstein, G. Z., Vue, T-L, Ohlstein, E. H., and
Douglas, S. A. Use of differentialdisplayto
identify differentially expressed
44. Laemmli,
U. K. Cleavage
of structural
proteins
during the assembly
the head of bacteriophage
T4. Nature (Lond.), 227: 680-685,
1970.
43. Autieri, M. V., Olson, B. A., Vue, T-L, and Feuerstein,
G. Z. Antisense
oligonucleotides
to the p65 subunit of NF-kB inhibit human vascular
smooth muscle cell adherence and proliferation.
Biochem.
Biophys.
Res.
Commun.,
213: 827-831,
1995.
of