Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2273-2283
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2273-2283.)
© 1995 American Heart Association, Inc.
Sodium Butyrate Inhibits Platelet-Derived Growth FactorInduced Proliferation of Vascular Smooth Muscle Cells
Kasturi Ranganna;
Trupti Joshi;
Frank M. Yatsu
From the Department of Neurology, University of Texas Health Science
Center at Houston (Tex).
Correspondence to Kasturi Ranganna, Department of Neurology, University of Texas Health Science Center at Houston, Houston, TX 77030.
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Abstract
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Abstract Sodium butyrate (SB), a naturally occurring
short-chain
fatty acid, was investigated for its therapeutic value
as an
antiproliferative agent for vascular smooth muscle cells (SMCs).
At
5-mmol/L concentration, SB had no significant effect on rat
SMC
proliferation. However, at the same concentration, SB inhibited
platelet-derived
growth factor (PDGF)-AA, -AB, and
-BBinduced
proliferation of SMCs. Exposure of SMCs to PDGF-BB
resulted
in activation of receptor intrinsic tyrosine kinase activity
and
autophosphorylation of ß-PDGFreceptor
(ß-PDGFR).
The activated ß-PDGFR physically associated and
phosphorylated
signaling molecules such as
ras-GTPase activating protein (GAP)
and phospholipase C

(PLC

). SB, in the absence of PDGF-BB, caused
neither ß-PDGFR
tyrosine phosphorylation nor
phosphorylation
and association of GAP and PLC

with
ß-PDGFR. PDGF-BBenhanced
activation of receptor intrinsic tyrosine
kinase activity and
autophosphorylation of tyrosine
residues of ß-PDGFR were
unaffected by SB irrespective of whether
SMCs were preincubated
with SB before exposure to PDGF-BB plus SB or
incubated concomitantly
with PDGF-BB plus SB. Likewise,
phosphorylation and association
of GAP and PLC

with
PDGF-BBactivated ß-PDGFR were
unaffected. In addition, SB
did not block PDGF-BBstimulated,
PLC

-mediated
production of inositol triphosphate. Similarly,
PDGF-BBinduced
ß-PDGFR degradation was unaffected when SMCs were
exposed
to PDGF-BB plus SB, and SB by itself had no influence on
ß-PDGFR
degradation. Unlike ß-PDGFR kinase activity,
mitogen-activated
protein kinase (MAP-kinase) activity was
stimulated by SB by
about 2.7-fold. Exposure of SMCs to PDGF-BB caused
an

11.4-fold
increase in MAP-kinase activity and this increase in
activity
was not significantly affected when cells were coincubated
with
PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with
SB
for 30 minutes and subsequent incubation in PDGF-BB plus
SB abolished
most of the PDGF-BBinduced MAP-kinase activity
(4.6-fold).
Transcription of growth response genes such as c-
fos,
c-
jun,
and c-
myc were induced by PDGF-BB, and
their induction was suppressed,
particularly c-
myc, by
incubating SMCs with PDGF-BB plus SB.
Similarly, preincubation of cells
with SB for 30 minutes and
subsequent incubation in PDGF-BB plus SB
diminished PDGF-BBinduced
transcription of c-
fos,
c-
jun, and c-
myc. However, SB by itself
had no
significant effect on c-
fos, c-
jun, and
c-
myc transcription.
Our data suggest that the inhibition of
PDGF-BBinduced
proliferation of SMCs by SB involves
MAP-kinaseregulated
events as well as transcription of
growth-response genes.
Key Words: sodium butyrate proliferation platelet-derived growth factor smooth muscle cells
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Introduction
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Atherosclerotic plaque
development involves several synchronized
events such as adhesion,
migration, proliferation, and transformation
of cells, and these events
are mediated by growth factors and
cytokines. A greater
attention has been focused on PDGF in the
origin of
atherosclerosis because of its
mitogenic,
1 2 chemotactic,
3 and
vasoconstrictor
4 effects on VSMCs. The implication that
VSMC
proliferation contributes to hypertension,
5 6
atherosclerosis,
7 8 and
restenosis after angioplasty
8 9 10 11 has led to a
growing
interest in the use of drugs to intercept this process.
SB, a natural bacterial fermentation product produced in mammalian
colon,12 has been shown to be a differentiation promoter
with a potent antiproliferative effect on several cancer
cells.13 14 15 16 17 18 19 In addition to its antiproliferative activity,
SB induces changes in cellular morphology,20 21 alters the
expression of cellular genes,22 23 24 25 26 27 28 29 30 31 and modulates hormone
action and hormone receptors23 25 26 27 28 29 30 31 as well as growth
factor receptors.31 32 SB has also been shown to inhibit
the high fat dietinduced mammary tumorigenesis.33
Additionally, SB has been used in preliminary clinical trials to treat
certain acute leukemias34 35 and stable butyrate
derivatives have been developed with a hope to use these compounds to
treat mammary carcinoma.36 Since atherosclerotic plaque
development involves proliferation of VSMCs, and SB is a naturally
occurring short-chain fatty acid, it prompted us to investigate the
potential therapeutic value of SB as an antiproliferative agent of
VSMCs. In the present study we report that SB inhibited
PDGF-induced proliferation of rat SMCs. We examined the effect of SB on
various aspects of SMC proliferation and signal transduction pathways
involved in the proliferative response to PDGF to understand the
mechanism of inhibition of PDGF-induced proliferation of SMCs by SB.
This information will provide clues to the step(s) that can be targeted
for intervention of atherosclerotic plaque development.
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Methods
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Materials
Recombinant PDGF-AA, -AB, and -BB were obtained from GIBCO BRL
Life
Technologies. SB came from Fluka. A7r5 rat aortic SMCs were
obtained
from American Type Culture Collection.
Anti-phosphotyrosine,
anti-ßPDGFR, anti-GAP, and anti-PLC

were purchased
from Upstate Biotechnology Inc. Anti-MAPkinase and
protein
A/G PLUS-agarose conjugate were from Santa Cruz Biotechnology.
[Methyl-
3H]thymidine,
Myo-[2-
3H]inositol,
and [

-
32P]ATP and reagents for ECL were
purchased from
Amersham Corp.
Cell Cultures
Rat SMCs were grown in DMEM fortified with 10% FCS, 50 U/mL
penicillin, and 50 µg/mL streptomycin. For proliferation assay, cells
were plated in six-well trays. Cells were seeded into
25-cm2 flasks for myo-[3H]inositol
incorporation studies. For other studies, cells were cultured in 150-mm
culture dishes. Cells were grown to 90% confluence at 37°C in a
humidified atmosphere of 5% CO2. Culture media were
changed every other day. Experiments were performed using 90%
confluent cells. Quiescent cells were obtained by incubating 90%
confluent cells either in 0.1% serum containing medium for 48 hours or
in serum-free medium for 24 hours. Different concentrations of
PDGF-AA, -AB, or -BB were used for DNA synthesis measurements. Since 30
ng/mL PDGF-BB results in maximal induction of IP3
production in rat VSMCs,37 the same concentration
was used in the present study for phosphoinositide
assay. For protein phosphorylation, protein
association, MAP-kinase, and gene transcription assays, 50 ng/mL
PDGF-BB was used. These are short-term-exposure studies
resulting in immediate early responses to PDGF-BB. To ensure efficient
response, maximum concentration of PDGF-BB needed for full induction of
SMC proliferation was used,
20 to 50 ng/mL. In addition, almost all
published signal transduction studies involving PDGF-BB used similar
concentrations for early response studies (40 to 60 ng/mL).
Concentration of SB used in the experiments was 5 mmol/L unless
otherwise noted.
Mitogenic Assay
Quiescent cells were stimulated with PDGF in a serum-free
medium containing 1 µCi/mL [3H]thymidine in the absence
or presence of SB. After 24 hours of incubation, the cells were washed
three times with PBS and fixed in 5% ice-cold TCA for 1 hour at
4°C. Fixed cells were washed three times with 5% ice-cold TCA
and dissolved in 0.2N NaOH/0.1% SDS. Amount of radioactivity
incorporated was measured.
Proliferation Assay
To measure proliferation, cells were grown to 90% confluence
and then made quiescent by incubation in 0.1% FCS containing DMEM
medium for 48 hours. Cells were then stimulated with serum-free
DMEM containing 10 ng/mL PDGF-BB in the absence or presence of SB.
After 24 hours of incubation, the cells were washed with PBS and
dissociated with trypsin/EDTA. Dissociated cells were counted in
triplicate with a hemocytometer. Cell viability was determined by
trypan blue exclusion. Cell viability was more than 90% up to 5
mmol/L, both in the presence and absence of PDGF-BB. Between 5 and 10
mmol/L SB concentration, cell viability was reduced to 60% to
70%.
Phosphoinositide Assay
Quiescent cells prelabeled with myo-[3H]inositol
(4 µCi/mL) for 48 hours were incubated in PBS containing 20 mmol/L
lithium chloride for 10 minutes. After 10 minutes,
phosphoinositide assay was initiated by the addition of
test compounds and terminated after 1 minute by addition of 2 mL of 5%
TCA. TCA supernatants collected by centrifugation were
extracted three times with ether and neutralized before separation of
inositol phosphates on anion exchange resin (AG 1X48; 200 to 400 mesh;
Formate form). IPs, IP2s, and IP3s were
separated according to the protocol described by Berridge et
al.38 Radioactivity in each of these inositol phosphates
was determined by liquid scintillation counting.
Immunoblots
Cells grown in 150-mm culture plates were washed with
ice-cold PBS three times and lysed in 1 mL of RIPA buffer (20
mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 10% glycerol, 0.1% SDS, 0.5%
sodium deoxycholate, 1% Triton X-100, 2 mmol/L EDTA, 1 mmol/L PMSF, 20
µmol/L leupeptin, and 1 mmol/L sodium orthovanadate) by gentle
rocking for 20 minutes at 4°C.39 40 Lysates were then
cleared by centrifugation at 10 000g for 15
minutes at 4°C. The clear lysates were normalized for total cellular
protein, and an equal amount of protein from each lysate was
fractionated on SDS-polyacrylamide gel. Polypeptides, separated
by SDS-PAGE, were transferred to immobilon PVDF membrane and blocked
for 1 hour in blocking buffer containing 5% nonfat dry milk and 0.1%
Tween 20 in TBS. Blots were washed three times with TBS containing
0.1% Tween 20 and probed with appropriate primary antibody for 1 hour
in TBS. Blots were washed three times with TBS containing 0.1% Tween
20 and then incubated with appropriate second antibody conjugated to
horseradish peroxidase. Immunoreactive proteins were identified using
an ECL detection kit from Amersham Corp.
Immunoprecipitation
Cell lysates were prepared as described above using RIPA
buffer.39 40 Equal amounts of proteins were incubated with
ß-PDGFR, GAP, PLC
, or P-Tyr antibody and 20 µL of protein A/G
PLUS agarose-conjugate for about 12 hours at 4°C.
Immunoprecipitated complexes were collected by
centrifugation and washed once with RIPA buffer, twice
with 0.5 mol/L LiCl in 0.1 mol/L Tris, pH 7.4, and once with 10 mmol/L
Tris, pH 7.4. Immunoprecipitated proteins were eluted with SDS-PAGE
buffer and separated on 7.5% gels. Separated proteins were transferred
to immobilon PVDF membrane and used for immunoblotting
as described above with suitable antibodies.
MAP-Kinase Assay
Cell lysates were prepared by using nonionic detergent buffer
containing 1% Triton X-100, 20 mmol/L Tris, pH 8.0, 137 mmol/L NaCl,
10% glycerol, 2 mmol/L EDTA, 1 mmol/L PMSF, 20 µmol/L leupeptin, 50
mmol/L sodium fluoride, and 1 mmol/L sodium orthovanadate as
described above.41 42 Similar amounts of proteins were
incubated with polyclonal MAP-kinase antibody (C-16, ERK1, Santa Cruz
Biotechnology) for 2 hours at 4°C, and 20 µL of protein A/G PLUS
agarose-conjugate was added for the last 30 minutes.
Immunocomplexes were collected and washed twice with nonionic detergent
buffer followed by one wash in TBS supplemented with 1 mmol/L sodium
orthovanadate and 5 mmol/L benzamidine. MAP-kinase activity was
measured by addition of 40 µL of kinase reaction mix (30 mmol/L Tris,
pH 8.0, 10 mmol/L MgCl2, 20 µmol/L ATP, 1 mmol/L
DTT, 5 mmol/L benzamidine, 10 µCi of [
-32P]ATP and 6
µg of MBP) and incubation for 30 minutes at 37°C.41 42
Reaction was terminated by addition of 20 µL of 4x SDS-PAGE buffer.
Reaction products were analyzed by SDS-PAGE on 12% gel
followed by autoradiography.
Phosphorylated MBP was excised from the dried gel, and
radioactivity was measured by Cerenkov counting.
RNA Isolation and Northern Analysis
Quiescent SMCs grown in 150-mm culture dishes were exposed to
different treatments for times indicated in the relevant
"Results" section. Total RNA was isolated using TRI reagent
method (Molecular Research Center). For Northern analysis, 15
µg of total RNA was denatured in sample loading buffer containing 1
µg/µL ethidium bromide and electrophoresed through 1% agarose
formaldehyde gel.43 RNA was transferred to S&S MaxS nytran
membrane by downward alkaline transfer method44 by using
S&S Turbo Blotter transfer system. Transferred RNA was fixed to nytran
membrane by ultraviolet irradiation. c-fos,
c-jun, and c-myc cDNAs were labeled by random
priming with the New England Biolabs NEBlot kit and
[32P]dCTP as recommended by the manufacturer. G3PDH
oligonucleotide probe was labeled at the 5' end by the
T4 polynucleotide kinasecatalyzed
transfer of
-phosphate from
[32P]ATP to the 5'
end of oligonucleotide. RNA blots were probed with
random primer labeled c-fos, c-jun,
c-myc, or 5' end-labeled 60-baselong G3PDH
oligonucleotide probe.45 Blots were
exposed to hyperfilm-MP (Amersham Corp) with intensifying screen at
-70°C for 1 to 3 days. The autoradiographs were scanned with a
BioRad GS-670 densitometer to quantify the intensity of hybridization
signals.
 |
Results
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Measurement of DNA Synthesis
Growth-arrested SMCs were exposed to PDGF-AA, -AB, or -BB with
or
without 5 mmol/L SB for 24 hours in the presence of
[
3H]thymidine.
After 24 hours, the amount of
[
3H]thymidine incorporated was
measured, and the results
are shown in Fig 1

. Although PDGF
is a potent mitogen
for SMCs, not all three isoforms are equally
potent. PDGF-AA is the
least potent and PDGF-BB is the most
potent of the three isoforms of
PDGF. Maximal mitogenic responses
to PDGF-AB and -BB were
approximately 5.3- and 6.2-fold above
unstimulated values. The
corresponding ED
50 values for the mitogenic
response
to PDGF-AB and -BB were 7 and 5 ng/mL, respectively. However,
stimulation
of [
3H]thymidine incorporation by PDGF-AA was
only about 2.5-fold
above unstimulated values at 20 ng/mL and remained
the same
above 20 ng/mL. SB at 5 mmol/L inhibited PDGF-AB and
-BBinduced
DNA synthesis by more than 90%. On the other hand,
PDGF-AAinduced
[
3H]thymidine incorporation was
inhibited by about 65%.
Since PDGF-BB is the most effective isoform of PDGF for SMCs, we
extended the study to investigate the effective concentration of SB on
PDGF-BBinduced proliferation (Tables 1
and 2
). Effect of SB on PDGF-BBinduced DNA synthesis
(Table 1
) and on increase in cell number (Table 2
) was measured between
the concentration of 1 mmol/L and 8 mmol/L. At 1 mmol/L SB there was
about 25% inhibition of PDGF-BBinduced DNA synthesis. Concentrations
of SB higher than 1 mmol/L exhibited significant inhibition of
PDGF-BBinduced DNA synthesis. At 5-mmol/L concentration, SB
completely inhibited PDGF-BBinduced DNA synthesis. To investigate
whether similar inhibitory effects of SB are observed on
PDGF-BBinduced increased cell number, we determined the number of
cells after 24 hours' exposure to different concentrations of SB in
the absence or presence of PDGF-BB (Table 2
). As shown in Table 2
,
PDGF-BB induced an increase in cell number, and this increase in cell
number was reduced significantly in the presence of SB irrespective of
concentrations used. However, at up to 5-mmol/L SB concentration, more
than 90% of the cells were viable in both the absence and presence of
PDGF-BB. At an 8-mmol/L concentration of SB, cell viability was reduced
to about 60% and 70% in the absence and presence of PDGF-BB,
respectively.
Although at a 5-mmol/L concentration PDGF-BBinduced DNA synthesis as
well as cell number was completely inhibited by SB, PDGF-BBstimulated
protein and RNA contents were reduced by only about 40% (Table 3
). In the absence of PDGF-BB, 5 mmol/L SB had no effect
on protein and RNA content as in unstimulated cells. Since a 5-mmol/L
concentration of SB almost completely inhibited PDGF-BBinduced DNA
synthesis and cell proliferation without any adverse effect on SMCs, we
selected this concentration of SB to extend our studies to understand
the mechanism of inhibition of PDGF-BBinduced proliferation.
PDGF-BB-Induced Protein Tyrosine
Phosphorylation
Protein tyrosine phosphorylation has been
implicated in mitogenic signal pathway. Therefore, we
probed into PDGF-BBstimulated protein tyrosine
phosphorylation and its response to SB treatment (Fig 2
). Although anti-Ptyr antibody recognized some bands
in all the treatments, it identified distinct proteins that are highly
phosphorylated on tyrosine residues on exposure to
PDGF-BB compared with untreated cells. Coincubation of cells with SB
and PDGF-BB caused no alteration in PDGF-BBenhanced protein tyrosine
phosphorylation profile. Similarly, pretreatment of
cells for 30 minutes with SB and subsequent exposure to PDGF-BB plus SB
had no influence on PDGF-BBinduced protein tyrosine
phosphorylation profile. On the other hand, SB by
itself caused no increased protein tyrosine
phosphorylation similar to untreated cells.

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Figure 2. Blot shows influence of SB on PDGF-BBinduced
protein tyrosine phosphorylation. Quiescent SMCs in
serum-free DMEM were exposed for 5 minutes with no addition (Lane
1), 5 mmol/L SB (Lane 2), preincubation in 5 mmol/L SB for 30 minutes
and then with 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 3), 50 ng/mL
PDGF-BB (Lane 4), or 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 5) at
37°C. After incubation, cells were lysed as described in
"Methods." Equal amounts of whole-cell proteins were
separated by electrophoresis on 7.5% SDS-polyacrylamide gels,
transferred to PVDF membrane, and immunoblotted with
anti-P-Tyr antibody. Immunoreactivity was detected by ECL.
PDGF-BBinduced tyrosine phosphorylation of cellular
proteins is indicated by the arrows.
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To assess whether PDGF-BBinduced ß-PDGFR autophosphorylation is
modulated by SB, we analyzed anti-PTyr immunoprecipitates by
immunoblotting with anti-ß-PDGFR antibody (Fig 3B
). As in cellular proteins, PDGF-BBinduced ß-PDGFR
tyrosine phosphorylation was unaffected by SB
irrespective of whether the cells were exposed to SB before PDGF-BB
plus SB or together with PDGF-BB. Cells exposed to no additions or SB
alone exhibited no ß-PDGFR tyrosine phosphorylation.
In addition, SB had no effect on ß-PDGFR level (Fig 3A
), and it was
similar irrespective of the treatment.

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Figure 3. Blots show effect of SB on
PDGF-BBactivated ß-PDGFR
autophosphorylation. Density-arrested cells
were treated with similar additives as described in legend to Fig 2 for
5 minutes at 37°C. Cell lysates were prepared and processed
for immunoprecipitation and immunoblotting as described
in "Methods." Similar amounts of cell lysates were used for
immunoprecipitation with anti-ßPDGFR or anti-PTyr. A,
Anti-ßPDGFR immunoblot of anti-ßPDGFR
immunoprecipitates. B, Anti-ßPDGFR immunoblot of
anti-PTyr immunoprecipitates.
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Association of Signaling Molecules with ß-PDGFR
PDGF treatment of cells has been shown to result in PDGFR
association and phosphorylation of signaling molecules
such as PLC
, GAP, raf-I, and PI3-kinase. To ascertain
whether inhibition of PDGF-BBinduced proliferation of SMCs by SB
involved disruption of this signal transducing event,
phosphorylation of GAP and PLC
and association of
phosphorylated GAP and PLC
with activated
ß-PDGFR were examined.
Although equal amounts of cell lysates were used for GAP
immunoprecipitation, immunoblotting with anti-GAP
antibody revealed that the amount of GAP immunoprecipitated was less in
unstimulated and SB-treated cells compared with PDGF-BBtreated cells
(Fig 4A
). PDGF-BBstimulated increase in GAP
immunoprecipitation was not altered by exposing SMCs to SB before the
addition of PDGF-BB plus SB or coincubation with PDGF-BB and SB. This
differential effect of anti-GAP antibody on GAP immunoprecipitation
suggests that the GAP antibody probably recognized
PDGF-BBactivated GAP more efficiently than unstimulated GAP,
as in no-addition control and in cells that were treated with SB
alone (Fig 4A
). To determine whether SB interferes with
PDGF-BBstimulated tyrosine phosphorylation of GAP,
similar amounts of cell lysates were immunoprecipitated with anti-P-Tyr
and immunoblotted with anti-GAP (Fig 4B
). It was evident
that SB did not alter PDGF-BBenhanced tyrosine
phosphorylation of GAP irrespective of whether the
cells were exposed to SB before the addition of PDGF-BB plus SB or
coincubated with PDGF-BB and SB. SB by itself caused no protein
tyrosine phosphorylation as in no-addition
treatment. Similar to tyrosine phosphorylation of GAP,
association of activated GAP with ß-PDGFR was not modified by
SB (Fig 4C
). Immunoblotting of GAP immunoprecipitates
with anti-ßPDGFR revealed that ß-PDGFR was associated with GAP
only in immunoprecipitates prepared from PDGF-BBtreated cells
irrespective of the presence or absence of SB.

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Figure 4. Blots show effect of SB on PDGF-BBstimulated
tyrosine phosphorylation of GAP and its association
with ß-PDGFR. Quiescent SMCs were incubated in serum-free DMEM
with similar additions as described in legend to Fig 2 . Equal amounts
of cell lysates were immunoprecipitated with anti-GAP or anti-P-Tyr and
processed for immunoblotting as detailed in
"Methods." A, Anti-GAP immunoblot of anti-GAP
immunoprecipitates. B, Anti-GAP immunoblot of anti-P-Tyr
immunoprecipitates. C, Anti-ß-PDGFR immunoblot of GAP
immunoprecipitates.
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Effect of SB on PDGF-BBinduced protein tyrosine
phosphorylation of PLC
and its association with
activated ß-PDGFR was investigated by using P-Tyr and PLC
immunoprecipitates, respectively (Fig 5
).
Immunoblotting of PLC
immunoprecipitates with
anti-PLC
revealed no significant effect on the levels of PLC
(Fig 5A
). As in GAP, PDGF-BB stimulated tyrosine
phosphorylation of PLC
that was not altered by SB
irrespective of whether cells were pre-exposed to SB before the
addition of PDGF-BB plus SB or coincubation with PDGF-BB and SB (Fig 5B
). In addition, association of activated PLC
with
ß-PDGFR was not modulated by exposing the cells to SB before the
addition of PDGF-BB plus SB or concurrent incubation with PDGF-BB and
SB (Fig 5C
). These observations suggest that the mechanism of
inhibition of PDGF-BBinduced SMC proliferation by SB does not involve
activation of ß-PDGFR and its interaction with GAP and PLC
.
Influence of SB on PDGF-BBStimulated IP3
Production
Exposure of SMCs to SB caused no increase in IP3
formation compared with no-addition control (Table 4
). Treatment of SMCs with PDGF-BB resulted in an
increase of about 30% in IP3 production, which was
increased to 60% by coincubating the cells with PDGF-BB and SB (Table 4
). This observation suggests that activity of PLC
was unaffected by
SB in the absence of PDGF-BB. But in the presence of PDGF-BB, SB
stimulated PDGF-BBinduced IP3 formation.
PDGF-BBInduced Downregulation of ß-PDGFR
To understand the mechanism of inhibition of PDGF-BBinduced
proliferation by SB, we also investigated the influence of SB on
PDGF-BBstimulated ß-PDGFR downregulation. Cell lysates obtained
from SMCs that were exposed to no-addition control, PDGF-BB, SB, or
PDGF-BB plus SB for different lengths of time were analyzed by
SDS-PAGE followed by immunoblotting with anti-ß-PDGFR
to study the receptor downregulation (Fig 6
). As shown
in Fig 6
, PDGF-BBinduced ß-PDGFR downregulation occurred after 30
minutes and was completely degraded after 2 hours of incubation of SMCs
with PDGF-BB. This time-dependent, PDGF-BBactivated
downregulation of ß-PDGFR was unaffected by SB when added together
with PDGF-BB. Cells exhibited a pattern of ß-PDGFR downregulation
similar to that in PDGF-BBtreated cells. Likewise, pretreatment of
cells for 30 minutes with SB and subsequent exposure to PDGF-BB plus SB
did not block PDGF-BBstimulated downregulation of ß-PDGFR (data not
shown). On the other hand, ß-PDGFR revealed no degradation when SMCs
were exposed to SB alone, indicating that SB did not interfere with
ligand-stimulated receptor downregulation.

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Figure 6. Blots show PDGF-BBinduced ß-PDGFR degradation.
Serum-starved SMCs were incubated in serum-free DMEM containing
no additions, 50 ng/mL PDGF-BB, 5 mmol/L SB, or 50 ng/mL PDGF-BB plus 5
mmol/L SB for indicated periods. Cell lysates were prepared and equal
amounts of cell lysates were subjected to PAGE as detailed in
"Methods." After electrophoresis, ß-PDGFR degradation was
detected by probing with anti-ßPDGFR antibody.
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PDGF-BBInduced MAP-Kinase Activity
MAP-kinases are serine and threonine kinases that are strongly
activated by PDGF. Effect of SB on PDGF-BBinduced MAP kinase
activity was measured by immunocomplex kinase assay. MAP-kinase was
immunoprecipitated from the SMC cell lysates by using anti-MAPkinase
antibody, and the immunocomplex was subjected to in vitro kinase
reaction by using MBP as a substrate (Fig 7
).
Surprisingly, exposure of SMCs to SB stimulated MAP-kinase activity by
about 2.7-fold over no-addition control. Addition of PDGF-BB to
SMCs stimulated MAP-kinase activity by about 11.4-fold. Coincubation of
SMCs with PDGF-BB plus SB caused no significant effect on
PDGF-BBstimulated MAP-kinase activity (10.3-fold). But preincubation
of SMCs with SB for 30 minutes and subsequent addition of PDGF-BB plus
SB resulted in a significant decrease in PDGF-BBinduced MAP-kinase
activity. There was only a 4.6-fold increase in activity over
no-addition control, suggesting that MAP-kinase activities are
differentially modified by SB.
Serum and PDGF-BBInduced Transcription of c-fos,
c-jun, and c-myc
Exposure of cells to serum or growth factors caused increased
rapid transcription of nuclear proto-oncogenes. We investigated the
effect of SB on serum and PDGF-BBstimulated c-fos,
c-jun, and c-myc transcription. The results of
these experiments are depicted in Fig 8
. Treatment of
serum-starved SMCs with serum caused increased transcription of all
three nuclear proto-oncogenes compared with no-addition
control. Serum-induced effect was more dramatic on c-fos
and c-jun than on c-myc. Addition of SB along
with serum diminished the serum-induced transcription of
c-fos (52%), c-jun (45%), and c-myc
(30%), although SB by itself had no significant effect on the
transcription of c-fos, c-jun, and
c-myc. Similar to serum, PDGF-BB also induced the
transcription of all three proto-oncogenes. SB diminished
PDGF-BBstimulated transcription of c-myc more strongly
than c-fos and c-jun. PDGF-BBinduced
transcription of c-myc was inhibited by about 70% to 80%,
whereas c-fos and c-jun transcription was
inhibited by about 45% irrespective of whether the cells were
preexposed to SB and then coincubated with SB plus PDGF-BB or treated
simultaneously with PDGF-BB plus SB.

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Figure 8. Blots show influence of SB on serum and
PDGF-BBinduced transcription of c-fos, c-jun,
and c-myc. Effect of SB on serum and PDGF-BBinduced
transcription of c-fos (A), c-jun (B), and
c-myc (C) is assessed at the time of their maximal
transcription (c-fos, 30 minutes; c-jun and
c-myc, 60 minutes). Serum-deprived SMCs in
serum-free DMEM were incubated with no addition (Lane 3), 10% FCS
(Lane 1), 10% FCS plus 5 mmol/L SB (Lane 2), 5 mmol/L SB (Lane 4),
preincubation in 5 mmol/L SB for 30 minutes and subsequent incubation
in 5 mmol/L SB plus 50 ng/mL PDGF-BB (Lane 5), 50 ng/mL PDGF-BB (Lane
6), or 50 ng/mL PDGF-BB plus 5 mmol/L SB (Lane 7) for 30 minutes or 60
minutes. After required incubation time, total RNA was isolated and the
levels of c-fos, c-jun, and c-myc
transcripts were measured as described in "Methods." The RNA
blots were probed with G3PDH to normalize the quantity of RNA loaded
onto each lane.
|
|
 |
Discussion
|
|---|
SB treatment of SMCs caused inhibition of PDGF-AA, -AB
and
-BBinduced proliferation. Although PDGF is a major
mitogen for many
cells, response to three PDGF isoforms seemed
to differ between cell
types. VSMCs responded to three PDGF
isoforms differently. In our
study, PDGF-AA was least mitogenic
of the three isoforms.
It stimulated about a 2.5-fold increase
in DNA synthesis whereas
PDGF-AB and -BB stimulated increases
in DNA synthesis of about 5.3-fold
and 6.2-fold, respectively.
These observations are in accordance
with previous reports.
37 46 47 It has been proposed that
differences in the mitogenic
sensitivity of the cells to
different PDGF isoforms are attributable
to both the absolute and
relative number of

-PDGFR and ß-PDGFR,
which differ depending on
cell types.
37 48 PDGF-AB and -BB
interacted with both

-
and ß-PDGFR. On the other hand,
PDGF-AA interacted exclusively with

-PDGFR. On the basis of
our DNA synthesis study, it appears that
SMCs expressed both

- and ß-PDGFR, but expression of ß-PDGFR was
higher
than

-PDGFR.
SB at a 5-mmol/L concentration inhibited SMC proliferation
induced by all three isoforms of PDGF, particularly PDGF-ABinduced
and -BBinduced proliferation. The antiproliferative effect of SB was
not mitogen-specific, because even serum-induced proliferation
of several mammalian cells was inhibited by SB.16 17 Other
studies have shown that insulin- and epidermal growth
factorinduced DNA synthesis is inhibited by SB.49
These reports indicate that SB is specific for the process of
proliferation rather than to the specific agent inducing proliferation.
The reason for studying the effect of SB on PDGF-induced SMC
proliferation is that PDGF is one of the key growth factors that induce
SMC proliferation. Proliferation of SMCs is the hallmark of
atherosclerosis.
Although the effective concentration of SB needed to inhibit
PDGF-BBinduced SMC proliferation is in millimoles per liter, up to 5
mmol/L SB concentration, no toxic effects were observed. Cell viability
was more than 90%. Moreover, the concentrations used in the
present study are probably close to the
physiological range.12 In addition, in
the absence of PDGF-BB, SB had no significant effect on baseline
proliferation (Tables 1
and 2
) as well as on cellular protein and RNA
levels (Table 3
). However, SB inhibited PDGF-BBinduced increase in
cellular protein and RNA level by about 40% in contrast to DNA
synthesis, which was completely inhibited. Since SB is an inducer of
differentiation and inhibitor of cell cycle
progression,15 50 51 it may be differently affecting the
expression of cell differentiation and proliferation-specific genes
in SMCs. Several reports indicate that SB can have different
consequences on the induction of specific RNAs in the same
cell.29 31 52 Previous studies have shown that the effects
of SB are caused by structural alteration in
chromatin15 53 54 as well as by regulation of
transcription regulatory proteins.55 56 SB has been shown
to cause hyperacetylation of histones15 53
and methylation of DNA,54 both of which may mediate SB
effect on gene expression. Hyperacetylation of histones
may lead to increased transcriptional activity, while DNA methylation
may cause inhibition of gene expression resulting in increase or
decrease in expression of specific genes depending on the status of the
cells. In our study it appears that some of the genes that were induced
by PDGF-BB were suppressed by SB, which may be
cell-proliferationrelated. Our unpublished data on G3PDH
indicate that it is one of the PDGF-BBinduced genes inhibited by SB.
Several other studies have shown increase in expression of
differentiation-specific genes in response to SB
treatment.22 23 24 25 26 27 28 29 30 31
How external growth signals carried by the growth factors are
transmitted from the cell surface receptors to the nucleus is still
vaguely understood. Substantial progress has been made in understanding
the initial steps, with the delineation of the structure of growth
factors and their receptor protein tyrosine kinases. PDGF, upon binding
to the receptor, causes conformational changes of the receptor leading
to receptor dimerization followed by activation of intrinsic tyrosine
kinase activity.57 58 59 As a result, PDGFR undergoes
autophosphorylation on tyrosines of the receptor
cytoplasmic domain, causing the activated receptor to associate
with signaling proteins that contain Src homology 2
domains60 61 such as PLC
,62 63 P1-3
kinase,64 65 raf-166 and
GAP.67 68 After activation and association with regulatory
signaling molecules, PDGFR was rapidly internalized and
degraded.65 69 70 71 We followed the effect of SB on these
initial steps of PDGF-induced signal transduction to delineate the mode
of action of SB. Our studies indicate that when SMCs are coincubated
with SB and PDGF-BB, SB not only has no effect on PDGF-BBstimulated
tyrosine phosphorylation of ß-PDGFR (Fig 3B
) but also
appears to have no significant effects on PDGF-BBinduced tyrosine
phosphorylation of cellular proteins resolved on the
Western blot (Fig 2
). Similarly, preincubation with SB and subsequent
exposure to PDGF-BB plus SB had no effect on protein tyrosine
phosphorylation. This observation suggests that SB
effect is independent of PDGF-BBinduced protein tyrosine
phosphorylation of ß-PDGFR. This observation is
further substantiated by phosphorylation and
association of signaling molecules such as GAP and PLC
with
ß-PDGFR (Figs 4
and 5
). Exposure of SMCs to no addition or to SB
caused no tyrosine phosphorylation or association of
proteins. In the presence of PDGF-BB whether it was added with SB or
subsequent to SB addition, ß-PDGFR
autophosphorylation and its association with GAP
and PLC
as well as their phosphorylation were
unaffected. This suggests that mechanism of inhibition of
PDGF-BBinduced SMC proliferation by SB is not through its effect on
PDGF-BB bindinginduced autophosphorylation on
tyrosines of the receptor cytoplasmic domains and its association with
signaling proteins GAP and PLC
.
Although there was no difference in the levels of ß-PDGFR (Fig 3A
)
and PLC
(Fig 5A
) in different treatments, GAP immunoprecipitates
exhibited differences in the levels of GAP (Fig 4A
). Same amounts of
proteins were used for immunoprecipitation of GAP from the cells
exposed to different treatments. Yet we noticed an increase in GAP
protein in GAP immunoprecipitates prepared from cells exposed to
PDGF-BB, irrespective of whether SB was present or not. Since GAP
undergoes tyrosine phosphorylation on exposure to
PDGF-BB, it may cause conformational change in GAP, which may result in
increased recognition by GAP antibody. Therefore, we observed an
increase in GAP protein in GAP immunoprecipitates when cells are
activated with PDGF-BB. Similar differences have been noted in
other reports.68
Effect of SB on PDGF-BB binding to ß-PDGFR was determined by
following ß-PDGFR downregulation. These studies revealed that SB has
no effect on PDGF-BBstimulated ß-PDGFR degradation. SMCs exhibited
similar time-dependent PDGF-BBinduced ß-PDGFR downregulation
both in the absence and presence of SB. On the other hand, SB by itself
did not react with ß-PDGFR and exhibited no ß-PDGFR degradation
(Fig 6
). These observations suggest that the site of SB action may be
downstream to the signaling pathway.
Analysis of IP3 reveals that SB by itself had no
effect on PLC
-catalyzed IP3 formation. However,
PDGF-BB stimulates IP3 formation. PDGF-BBinduced
IP3 formation was not inhibited by exposing the SMCs to
PDGF-BB plus SB. In fact, there was increased stimulation of
IP3 formation when the cells were coincubated with PDGF-BB
and SB than when they were exposed only to PDGF-BB. Although we do not
know the mechanism by which PDGF-BB plus SB increased IP3
formation, there is an additional pathway that causes IP3
formation by PLC. This pathway was mediated by guanosine
nucleotide protein, referred to as Gp.72 It is
suggested that there was some form of interaction between PDGFR and Gp
protein that caused an increase in IP3 formation from
phosphatidylinositol 4,5-biphosphate by PLC. Increase in
PDGF-BBinduced IP3 in PDGF-BB plus SBtreated cells was
probably due to PLC activated by G-proteinmediated
pathway. However, at the moment the significance of induction of
IP3 in PDGF-BB plus SBtreated cells is not clear.
Hydrolysis of phosphoinositide by PLC
released two
second messengers: diacylglycerol, an activator of protein
kinase C, and IP3, a generator of calcium signal, by
releasing calcium from intracellular stores.73 74
Intracellular calcium is known to regulate biochemical processes either
directly or indirectly through specific calcium binding
proteins.75 However, we do not know whether calcium
binding proteins have any role in SB-inhibited, PDGF-BBinduced SMC
proliferation.
A key factor in the signaling pathway involved in transducing the
signal from an activated protein tyrosine kinase to an
intracellular response has been identified as the family of
MAP-kinases. MAP-kinases, which are also called extracellular
signal-regulated kinases (ERKs), are serine/threonine kinases.
Although MAP-kinase 1 and 2 are the most extensively studied
MAP-kinases, several other isoforms of MAP-kinase have recently been
identified.76 MAP-kinases phosphorylate and
regulate the activity of enzymes and transcription
factors.77 Our studies revealed that SB stimulates
MAP-kinase activity by about 2.7-fold compared with uninduced control
cells. However, it is less than the MAP-kinase activity induced by
PDGF-BB (11.4-fold). This PDGF-BBstimulated activity is not
significantly affected when cells are simultaneously
exposed to PDGF-BB plus SB (10-fold). But the PDGF-BBstimulated
MAP-kinase activity is significantly inhibited if cells are preexposed
to SB for 30 minutes before treatment with PDGF-BB plus SB, suggesting
that preincubation with SB is blocking the activity that is
activated by PDGF-BB. MAP-kinases are activated by
concurrent phosphorylation of tyrosine and threonine
residues. This double phosphorylation is mediated by
the dual specificity MAP-kinase kinase (MKK or MEK).78 MEK
is in turn regulated through phosphorylation by
MAP-kinase kinase kinases (MEKK), which include the proto-oncogene
product raf.79 In addition, MAP-kinase can
undergo autophosphorylation on tyrosine
residues.80 It is reasonable to think that the small
amount of activation of MAP-kinase activity induced by SB may be the
result of autophosphorylation of tyrosine residues.
Similar induction of MAP-kinase activity was observed by Cook and
McCormick.42 Protein phosphorylation and
dephosphorylation are essential mechanisms in the
pathways by which cell proliferation is regulated. For full activation
of MAP-kinases, one or more distinct protein kinases are needed, and
increased induction of MAP-kinase activity in PDGF-BBtreated cells
could be due to activation of one or more of these distinct protein
kinases. At the moment we do not know the mechanism of inhibition of
MAP-kinase activity in cells that are pretreated with SB before
treatment with PDGF-BB plus SB. It can be due to inactivation of a
kinase that is supposed to activate MAP-kinase (MEK or MEKK?)
or it can be due to the stimulation of a phosphatase that blocks the
activation of MAP-kinase. Although MAP-kinase can be completely
inactivated in vitro by treatment with either CD-45, a
phosphotyrosine-specific phosphotyrosine phosphatase, or the
catalytic subunit of phosphatase 2A, a predominantly
serine/threonine-specific protein phosphatase, the mechanism of
MAP-kinase inhibition in intact cells is not understood.81
But recently it has been reported that a growth-induced gene MKP1
encodes a dual specificity phosphatase that
dephosphorylates and inactivates MAP-kinase
more specifically than other phosphatases.82 Understanding
of these phosphatases may provide clues to the mechanism of inhibition
of MAP-kinase by SB.
One of the earliest genetic changes elicited by many mitogens is the
induction of expression of immediate early growth-response
genes.83 84 These genes couple early biochemical
second-messenger signals to long-term changes in transcription,
resulting in mitogenesis. Proto-oncogenes such as c-fos,
c-jun, and c-myc are some of the immediate early
growth-response genes that are induced in response to PDGF
treatment.81 85 86 87 In our study SB inhibited
PDGF-BBstimulated c-fos, c-jun, and
c-myc transcription irrespective of whether SMCs were
preexposed to SB, before treatment with PDGF-BB plus SB, or treated
concurrently with PDGF-BB plus SB. But SB inhibited PDGF-BB
upregulated c-myc expression more severely than
c-fos and c-jun expression. The reason for
differences in extent of inhibition of PDGF-BBstimulated expression
of c-fos, c-jun, and c-myc is not
clear. However, it is interesting that extent of inhibition of
c-fos and c-jun expression by SB was similar. The
importance of proto-oncogenes lies in the fact that they
activate or suppress second-phase genes essential for
cellular proliferation by interacting with their respective promoter
regions. Since SB blocked the PDGF-BBstimulated expression of
proto-oncogenes, particularly c-myc, they may no longer
function as effective nuclear signal transducers to bring about a
cascade of gene-protein interactions that are manifested in
cellular proliferation. Downregulation of c-myc expression
has been observed in rectal carcinoma cells that were treated with
SB.88 89 In these cells the inhibitory effect
of SB on c-myc expression has been shown to be blocked by
inhibitors of protein synthesis, suggesting that SB causes
the synthesis of a protein(s) that downregulates c-myc
expression.88 Although the mechanism of inhibition of
c-myc by SB was not determined in our study, it probably
involves a trans mechanism as in rectal carcinoma cells.
However, at present we are investigating whether the effect of SB
on PDGF-BBinduced c-myc upregulation is at transcriptional
or posttranscriptional level. A considerable amount of constitutive
expression of c-myc was observed in cells maintained in
serum-free medium. Similar observations have been reported by
others, and it is suggested that constitutive c-myc may
represent a pool of functionally inactive, more stable,
nonpolyadenylated molecules.90 91
Taken together, our findings reveal that SB has a potential
antiatherosclerotic activity by suppressing PDGF-induced SMC
proliferation. Elucidation of the antiproliferative effect of SB would
greatly contribute to our understanding of the relationship between
cell proliferation and atherosclerosis.
 |
Selected Abbreviations and Acronyms
|
|---|
| DMEM |
= |
Dulbecco's modified Eagle's medium |
| ECL |
= |
enhanced chemiluminescence |
| ERKs |
= |
extracellular signal-regulated kinases |
| FCS |
= |
fetal calf serum |
| G3PDH |
= |
glyceraldehyde-3-phosphate dehydrogenase |
| GAP |
= |
ras-GTPase activating protein |
| Gp |
= |
guanosine nucleotide protein |
| IP |
= |
inositol monophosphate |
| IP2 |
= |
inositol biphosphate |
| IP3 |
= |
inositol triphosphate |
| MAP |
= |
mitogen-activated protein |
| MBP |
= |
myelin basic protein |
| MEK |
= |
MAP-kinase kinase |
| MEKK |
= |
MAP-kinase kinase kinases |
| MKK |
= |
MAP-kinase kinase |
| PBS |
= |
phosphate-buffered saline |
| PDGF |
= |
platelet-derived growth factor |
| PDGFR |
= |
platelet-derived growth factor receptor |
| PI3-kinase |
= |
phosphatidylinositol 3-kinase |
| PLC |
= |
phospholipase C |
| PMSF |
= |
phenylmethylsulfonyl fluoride |
| P-Tyr |
= |
phosphotyrosine |
| RIPA |
= |
radioimmunoprecipitation assay buffer |
| SB |
= |
sodium butyrate |
| SDS |
= |
sodium dodecyl sulfate |
| SDS-PAGE |
= |
SDS-polyacrylamide gel electrophoresis |
| SMC(s) |
= |
smooth muscle cell(s) |
| TBS |
= |
Tris-buffered saline |
| TCA |
= |
trichloroacetic acid |
|
 |
Acknowledgments
|
|---|
This work was supported by a grant from The Clayton Foundation
for
Research, Houston, Tex. We thank Dr D.S. Loose-Mitchell for
c-
fos,
c-
jun, and c-
myc probes, Dr P.
Dash for G3PDH probe, and Elaine
Marsh for the preparation of this
manuscript.
Received January 11, 1995;
accepted October 2, 1995.
 |
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