Ets-1 is an early response gene
activated by ET-1 and PDGF-BB in vascular smooth muscle
cells
Shinji
Naito1,
Shunichi
Shimizu2,
Shigeto
Maeda1,
Jianwei
Wang1,
Richard
Paul2, and
James A.
Fagin1,2
1 Division of Endocrinology and
Metabolism and 2 Department of
Molecular and Cellular Physiology, College of Medicine, University of
Cincinnati, Cincinnati, Ohio 45267-0547
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ABSTRACT |
Ets-1 is a transcription factor
that activates expression of matrix-degrading proteinases such as
collagenase and stromelysin. To study the control of
ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to
regulate VSMC migration and proliferation. Platelet-derived growth
factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of
ets-1 mRNA. These effects were
abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic
PMA treatment. Ets-1 mRNA was
superinduced by PDGF-BB and ET-1 in the presence of
cycloheximide. The chelation of intracellular
Ca2+ by
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester and the depletion of endoplasmic reticulum
intracellular Ca2+
concentration
([Ca2+]i)
by thapsigargin inhibited PDGF-BB- and ET-1-induced
ets-1 mRNA, whereas ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid had no effect. However,
[Ca2+]i
release alone was not sufficient to increase
ets-1 mRNA. Forskolin blocked ET-1-,
PDGF-BB-, and PMA-induced ets-1 mRNA,
as well as inositol phosphate formation, consistent with an effect
through impairment of PKC activation. Inhibitors of
ets-1 gene expression, such as H-7 and
herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We
propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of
vascular remodeling after arterial injury.
gene expression; thapsigargin; collagenase I; endothelin-1; platelet-derived growth factor
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INTRODUCTION |
THE PROTOONCOGENE
c-ets is the cellular progenitor of
v-ets and codes for a transcription
factor that activates the oncogene responsive unit of the polyoma virus
enhancer and interacts with the promoters of several eukaryotic genes
(28). The v-ets was originally
identified as a second transforming-specific sequence fused to the
v-myb oncogene in the avian leukemia
retrovirus E26. The c-ets-1,
ets-2,
erg,
elk-1, and
elk-2 genes belong to a family of
transcription factors that play an important role in the regulation of
cell proliferation and differentiation (4). The
c-ets-1 gene product cooperates with
the
c-fos/c-jun
complex [activator protein 1 (AP-1)] to activate expression
of certain promoters (28). Furthermore, the
ets-1 binding site, containing a
central GGAA/T core motif (PEA3), has been identified in the promoter of some matrix-degrading metalloproteinase genes such as stromelysin, collagenase, and urokinase plasminogen activator, suggesting possible roles for ets-1 in the regulation of
matrix degradation and tissue remodeling (10, 15).
After balloon intra-arterial injury, a program is initiated that
results in vascular smooth muscle cell (VSMC) migration, proliferation,
and a change in the composition and structure of the vascular
extracellular matrix (20). The signaling process that orchestrates the
injury response is multifactorial and involves a number of mesenchymal
cell growth factors, some of which are released from platelets adhering
at the injury site, whereas others are produced within the vessel wall
itself. In this vascular remodeling process, matrix-degrading
metalloproteinases are believed to be necessary for smooth muscle cell
(SMC) proliferation and migration into the neointima (3).
Ets-1 is expressed by periluminal
arterial SMC, with a first peak occurring 2 h after balloon injury
(12).
Serum induces ets-1 expression in
quiescent VSMC with a time course comparable to that seen after
arterial injury in vivo (12). The objective of this study was to
determine which factor, among the growth and chemotactic factors
believed to play a role in the early stages of the arterial response to
balloon denudation, stimulates the expression of this transcription
factor and to explore the signal transduction pathways involved.
We report that platelet-derived growth factor-BB (PDGF-BB) and
endothelin-1 (ET-1) are potent activators of
ets-1 gene expression in VSMC.
Furthermore, protein kinase C (PKC) activation and release of
Ca2+ stored in the endoplasmic
reticulum (ER) play an essential role in
ets-1 gene expression induced by
PDGF-BB and ET-1, which is not abrogated by inhibition of protein
synthesis. Induction of ets-1-mediated
transcriptional activity may be a common mechanism by which certain
growth and/or chemotactic factors activate a program of
extracellular matrix remodeling in vascular smooth muscle.
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EXPERIMENTAL PROCEDURES |
Cell culture.
VSMC were isolated from the aorta of 6-mo-old Sprague-Dawley rats
(Harlan Sprague Dawley, Indianapolis, IN) by digestion in 0.2% type I
collagenase (Sigma Chemical, St. Louis, MO) as described by
Chamley-Campbell et al. (5), with minor modifications. The cells were
then seeded in 75-cm2 plastic
flasks (Costar, Cambridge, MA; 3- 5 × 106 cells/flask) and grown in
Dulbecco's modified Eagle's medium-F-12 supplemented with 2.438 g/l
sodium bicarbonate, 50 µl/ml streptomycin and penicillin, and 10%
newborn calf serum at 37°C in 5%
CO2 in air. Cells were passaged by
treatment with 0.25% trypsin-0.02% EDTA in phosphate-buffered saline
(PBS). The cells used in the experiments were in their second or third
passage. All cell culture media and sera were purchased from GIBCO
(Grand Island, NY).
Reagents and cDNA probes.
PDGF-BB, insulin-like growth factor I (IGF-I), transforming growth
factor-
(TGF-
), and basic fibroblast growth factor (bFGF) were
purchased from Austral Biologicals (San Ramon, CA). ET-1 was from
Bachem Bioscience (King of Prussia, PA). Herbimycin A, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), phorbol 12-myristate 13-acetate (PMA), forskolin, thapsigargin, and ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA) were purchased from Sigma Chemical.
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) was from Molecular Probes (Eugene,
OR). The following cDNA probes were used: the rat
ets-1 probe was a 1.4-kilobase (kb)
BamH I fragment of
ets-1 cDNA cloned in pLXSN plasmid
vector; the human collagenase probe was a 2.2-kb Xho I fragment of p35-1 (American
Type Culture Collection 79062). For loading controls, blots were
rehybridized with a 1.4-kb Pst I
fragment of rat glyceraldehyde-3-phosphate dehydrogenase cDNA. Probes
were gel purified and 32P-labeled
by random priming as described previously (7).
Northern blot hybridization.
Total RNA was extracted from cultured rat VSMC with the TRIzol reagent
(GIBCO), according to the manufacturer's instructions. Twenty
micrograms of total RNA were size separated by electrophoresis through
1% agarose-formaldehyde gels. RNA samples were transferred to nylon
membranes (Micron Separation, Westborough, MA) and hybridized to the
indicated random prime-labeled cDNA probe. Hybridization reactions were
carried out for 16-24 h at 65°C in 0.25 M
Na2HPO4 (pH 7.2), 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS), and 30% formamide. Membranes were washed in 20 mM
Na2HPO4
(pH 7.2), 1 mM EDTA (pH 8), and 1% SDS. All experiments were performed
at least three times. Blots were exposed to Kodak X-Omat AR film at
80°C for 12-72 h and/or quantified with a
PhosphorImager (Molecular Dynamics).
Inositol phosphate formation.
Total inositol phosphate formation was measured essentially as
described previously (1, 2). Briefly, VSMC were prelabeled with 2.4 µCi/ml [3H]inositol
(Amersham, Arlington Heights, IL) for 24 h in inositol-free medium
containing 10% dialyzed fetal calf serum. VSMC were then washed and
incubated in PBS containing 20 mM
Li2+ with the indicated agents at
37°C for 5 min. Metabolic activity was stopped by addition of 100 µl of 10% perchloric acid and 10 µl phytic acid (20 mg/ml) on ice
for 20 min followed by centrifugation. The supernatants were
neutralized with 2 M KOH and 1 mM EDTA and subjected to anion exchange
chromatography. The final eluant was dissolved in scintillation fluid
and counted.
Measurement of intracellular
Ca2+
concentration.
Intracellular free Ca2+ was
assessed using the Ca2+-sensitive
fluorescent dye fura 2 (Sigma) according to the method of Grynkiewicz et al. (9). Cells were seeded in 35-mm-bottom glass dishes. After they
were plated, cells were incubated in 2 ml of complete culture medium
containing 5 µM fura 2-AM and 0.05% cremophor for 60 min at 37°C
under 5% CO2-95% air. The cells
were washed three times in a 3-(N-morpholino)propanesulfonic
acid (MOPS)-buffered physiological saline solution with a composition
of (in mmol/l) 140 NaCl, 4.7 KCl, 1.2 NaH2PO4,
20 MOPS, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, and 11 glucose (pH 7.4) and
allowed to equilibrate for 15 min at room temperature with or without
the indicated reagents. The fluorescence images of the cells were
recorded with a video image analysis system (InCa system, Intracellular
Imaging). Fluorescence intensities were not corrected for background,
as in control experiments autofluorescence of these cells was
negligible. The cells were excited sequentially at 340 and 380 nm, and
emissions at 510 nm were measured. The 340- and 380-nm images were
ratioed on a pixel-by-pixel basis, and the ratios were converted to
Ca2+ concentrations using a
previously generated standard curve. Standard solutions of free
Ca2+ (0-10 mM
Ca2+-EGTA, 100 mM KCl, and 10 mM
MOPS, pH 7.2; calcium calibration buffer kit II, Molecular Probes)
containing 10 µM fura 2 were used to make the standard curve. All
Ca2+ measurements were carried out
at room temperature.
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RESULTS |
Effect of growth and chemotactic factors on ets-1 mRNA levels.
As a first approach to examine the mechanisms controlling
ets-1 gene expression in VSMC,
serum-deprived cells were treated with selected growth or chemotactic
factors believed to play a role in VSMC proliferation and/or migration.
Figure 1 shows that serum
induced a concentration-dependent induction of
ets-1 mRNA. In addition, PDGF-BB,
ET-1, and PMA induced expression of a major ets-1 mRNA transcript of 5.3 kb and of
minor bands of 4.0 and 2.5 kb at 2 h after exposure (by ~7-fold). In
contrast, IGF-I, TGF-
, and bFGF had only a modest effect (1.8-, 2.2-, and 2.2-fold respectively) through a range of concentrations
(1-100 ng/ml, not shown). Notably, all factors increasing
ets-1 mRNA abundance are known to
activate PKC. In contrast, IGF-I and TGF-
signal through alternative
pathways, whereas the signaling intermediates for bFGF in SMC are
unclear. Activation of adenylyl cyclase activity by treatment with
forskolin appeared to decrease basal levels of
ets-1 mRNA (Fig. 1). The effects of
ET-1 and PDGF-BB on ets-1 mRNA were
maximal at 2 h (Figs. 2 and 5) and were
concentration dependent (Fig. 3).

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Fig. 1.
Effect of growth and chemotactic factors on
ets-1 mRNA abundance. Vascular smooth
muscle cells (VSMC) were serum starved for 48 h and then treated with
or without 5% serum, 10% serum, platelet-derived growth factor-BB
(PDGF-BB; 25 ng/ml), insulin-like growth factor I (IGF-I; 50 ng/ml),
transforming growth factor- (TGF- ; 5 ng/ml), basic fibroblast
growth factor (bFGF; 50 ng/ml), endothelin-1 (ET-1; 100 ng/ml), phorbol
12-myristate 13-acetate (PMA; 100 ng/ml), or forskolin (100 µM) for 2 h before RNA extraction. After electrophoresis of 20 µg RNA per
sample and transfer to a nylon membrane, the blot was sequentially
hybridized with 32P-labeled
ets-1 cDNA
(top) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA (bottom).
Arrows indicate ets-1 mRNA transcripts
of ~5.3, 4, and 2.5 kilobases (kb). Size of GAPDH is ~1.4
kb.
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Fig. 2.
Time course of ET-1 induction of ets-1
mRNA. VSMC were serum starved for 48 h and then treated with either
vehicle (control) or ET-1 (100 ng/ml) for indicated times.
Representative Northern blot of 20 µg RNA was sequentially hybridized
to either ets-1
(top) or GAPDH cDNA
(bottom) probes. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.
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Fig. 3.
Dose response of PDGF-BB and ET-1 induction of
ets-1 mRNA. Serum-starved VSMC were
treated with vehicle or indicated concentrations of PDGF-BB or ET-1 for
2 h. Northern blotting was as described in Fig. 1. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.
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Role of PKC activation on the regulation of ets-1 mRNA.
PMA activates PKC through binding to the
C1 region of its regulatory domain
(18). PMA stimulation by itself caused a significant increase in
ets-1 mRNA: it was maximal at 2 h and
returned completely to basal levels after 12 h (data not shown). Cells
were pretreated with PMA (200 ng/ml) for 24 h to deplete PKC activity.
The downregulation of PKC isozymes by chronic PMA treatment almost
completely abrogated the induction of
ets-1 mRNA stimulated by PDGF-BB,
ET-1, and PMA (Fig.
4A). H-7
inhibits PKC activity via a direct interaction on the catalytic site of
the enzyme and suppresses PKC-mediated phosphorylation. Preexposure to
20 µM H-7 attenuated the maximal ets-1 mRNA levels induced by PDGF-BB
(to 50% of stimulated levels), ET-1 (43%;
P = 0.07), and PMA (10%,
P < 0.01) (Fig.
4B). In contrast, herbimycin A, a
protein tyrosine kinase inhibitor, blunted the ets-1 mRNA stimulation by ET-1 (to 6%
of stimulated levels; P < 0.01) but
had a marginal effect on PDGF-BB- or PMA-induced
ets-1 mRNA (Fig.
4C).

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Fig. 4.
Effects of inhibition of protein kinase C or protein tyrosine kinase
activity on the regulation of ets-1
mRNA. A: VSMC were serum deprived for
24 h and then treated with or without 200 ng/ml of PMA for a further 24 h, before addition of one of the following agonists: PDGF-BB (25 ng/ml), ET-1 (100 ng/ml), or PMA (100 ng/ml) for 2 h.
B: VSMC were serum deprived for 48 h.
Cells were then pretreated with or without 20 µM H-7 for 1.5 h before
addition of the indicated agonists for 2 h.
C: VSMC were serum deprived for 48 h.
Cells were then pretreated with or without herbimycin A (0.75 µg/ml)
for 18 h before addition of the indicated agonists for 2 h. Northern
blotting was as described in Fig. 1. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb (and 4 kb, second arrow from top in
B) and GAPDH of 1.4 kb.
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Inhibition of protein synthesis is associated with superinduction of
ets-1 mRNA.
Activation of PKC results in the rapid stimulation of biosynthesis of
the c-Jun and c-Fos proteins, which after assembling into the AP-1
complex modulate the transcription of a repertoire of genes. Because
the ets-1 promoter contains a putative
AP-1 binding site, we examined whether general inhibition of protein synthesis (and hence of generation of AP-1 proteins) would impair ET-1
or PDGF-BB induction of ets-1 mRNA
content. Pretreatment with cycloheximide alone was associated with a
gradual accumulation of ets-1 mRNA
(Fig.
5A). In
the presence of the protein synthesis inhibitor, ET-1 and PDGF-BB
superinduced ets-1 mRNA content (Fig. 5, A and
B). The pattern of superinduction
was similar to that of c-jun, an
immediate early response mRNA that is not dependent on newly generated
transcription factor synthesis for expression. Thus
ets-1 gene expression in VSMC is
consistent with that of an immediate early response mRNA and does not
appear to be dependent on AP-1 synthesis.

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Fig. 5.
Effect of inhibition of either protein synthesis or transcriptional
activity on the regulation of ets-1
mRNA by ET-1 (A) or PDGF-BB
(B). VSMC were serum deprived for 48 h. One hour before stimulation with agonists, cells were treated with
or without 10 µg/ml cycloheximide (Cx) or 5 µg/ml actinomycin D
(AcD). Cells were harvested at indicated times after addition of ET-1,
PDGF-BB, or vehicle. Northern blots were sequentially hybridized with
the indicated cDNAs. Arrows point to
ets-1 mRNA transcripts of ~5.3 and
2.5 kb, c-jun mRNA of 2.7 kb, and
GAPDH of ~1.4 kb.
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Inhibition of ets-1 mRNA by adenosine 3',5'-cyclic
monophosphate-mediated signal transduction.
Forskolin stimulates the production of adenosine
3',5'-cyclic monophosphate (cAMP) and leads to the
activation of protein kinase A (PKA). There is ample documentation that
activation of PKA impairs phospholipase C (PLC)-mediated signal
transduction. Forskolin (100 µM) markedly inhibited
ets-1 mRNA induction by PDGF-BB, ET-1,
or PMA (Fig. 6). As shown in Fig.
7, addition of either ET-1 or PDGF-BB
significantly increased inositol phosphate generation. Pretreatment
with forskolin abrogated the ET-1- and PDGF-BB-stimulated inositol
phosphate production (Fig. 7), but, in these experimental conditions,
pretreatment did not appear to impair intracellular
Ca2+ mobilization (see Fig. 10).

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Fig. 6.
Effect of forskolin on agonist-stimulated
ets-1 mRNA expression. VSMC were serum
deprived for 48 h. Cells were treated with or without 100 µM
forskolin 5 min before addition of vehicle, PDGF-BB, ET-1, or PMA for 2 h. Northern blotting was as described in Fig. 1. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.
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Fig. 7.
Effect of forskolin on ET-1 or PDGF-BB stimulation of inositol
phosphate formation. VSMC were treated with or without 100 µM
forskolin for 5 min before addition of 100 ng/ml ET-1
(A) or 25 ng/ml PDGF-BB (B) for 1 min. Levels of inositol phosphates were determined as described in
EXPERIMENTAL PROCEDURES. Values are
expressed as means ± SD (n = 3 separate experiments). cpm, Counts/min.
* P < 0.05.
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Role of intracellular
Ca2+
concentration on the regulation of ets-1 mRNA.
A primary consequence of inositol 1,4,5-trisphosphate
(IP3) formation is the
stimulation of IP3-sensitive
Ca2+ channels in the ER and
release of Ca2+ stores. To explore
the role of intracellular Ca2+
concentration
([Ca2+]i)
on regulation of ets-1 gene
expression, cells were treated with either EGTA, a potent chelator of
extracellular Ca2+, or the
[Ca2+]i
chelator BAPTA-AM (19). Pretreatment with EGTA did not modify basal,
ET-1-, PDGF-BB-, or PMA-induced ets-1
mRNA levels. In contrast, [Ca2+]i
chelation with BAPTA-AM resulted in almost complete abrogation of
ets-1 mRNA production in response to
these stimuli (Fig. 8). Acute treatment
with thapsigargin, a selective inhibitor of the ER
Ca2+-ATPase, results in an
increase in
[Ca2+]i,
followed by sustained inhibition of
Ca2+ uptake and mobilization (see
Fig. 10). Interestingly, thapsigargin was not associated with an
increase in ets-1 mRNA, indicating that release of
[Ca2+]i
was not sufficient by itself to induce
ets-1 gene expression. However,
pretreatment with thapsigargin for 8 h to deplete intracellular Ca2+ content of the ER markedly
inhibited the induction of ets-1 mRNA by PDGF-BB, ET-1, and PMA (Fig. 9).

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Fig. 8.
Effect of EGTA and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester (BAPTA-AM) pretreatment on agonist-stimulated
ets-1 mRNA expression. VSMC were serum
deprived for 48 h. EGTA (5 mM) pretreatment was performed at 5 min
before addition of vehicle, PDGF-BB, ET-1, or PMA for 2 h. BAPTA-AM (30 µM) pretreatment was initiated 30 min before addition of agonists.
Northern blotting was as described in Fig. 1. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.
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Fig. 9.
Effect of thapsigargin (Tg) pretreatment on agonist-stimulated
ets-1 mRNA expression. VSMC were serum
deprived for 48 h and treated with either 100 nM thapsigargin or
indicated agonists for 2 h. To examine the effects of chronic exposure
to the endoplasmic reticulum
Ca2+-ATPase inhibitor,
serum-starved cells were pretreated with 100 nM thapsigargin for 8 h
before addition of vehicle or indicated agonists. Northern blotting was
as described in Fig. 1. Arrows point to
ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.
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The effects of ET-1 and effects of various inhibitors of
ets-1 gene expression on
[Ca2+]i
mobilization of fura 2-loaded SMC are shown in Fig.
10. Treatment with ET-1 caused a
transient
[Ca2+]i
increase within 5 s. Addition of 100 µM forskolin alone did not
modify
[Ca2+]i
levels (data not shown). Furthermore, pretreatment with forskolin did
not measurably alter
[Ca2+]i
release by ET-1 (Fig. 10B).
Similarly, 24-h treatment with PMA did not impact on the ET-1 effect on
[Ca2+]i
(Fig. 10C). The ET-1-induced release
of
[Ca2+]i
was only modestly impacted by a 5-min pretreatment with EGTA, although
[Ca2+]i
levels returned more rapidly to the baseline (Fig.
10D). Chelation of
[Ca2+]i
with BAPTA-AM almost completely prevented the ET-1-induced increase in
[Ca2+]i
(Fig. 10E). As discussed above,
treatment with thapsigargin evoked a gradual and sustained release of
[Ca2+]i
(Fig. 10F). However, after cells had
been exposed to thapsigargin for 8 h, they were no longer capable of
releasing
[Ca2+]i
in response to an acute challenge of ET-1 (Fig.
10G). At the concentrations used in
this study, thapsigargin did not impair phorbol ester- or ET-1-mediated
translocation of PKC-
and PKC-
, two of the most abundant PKC
isozymes in VSMC, as determined by Western blotting (data not shown).
This suggests that the ER
Ca2+-ATPase inhibitor does not
exhibit nonspecific upstream effects on the activation of PKC.

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Fig. 10.
Effect of forskolin, PMA, EGTA, BAPTA-AM, or thapsigargin on
ET-1-induced intracellular Ca2+
concentration mobilization in VSMC. Serum-starved cells were grown on
coverslips and loaded with fura 2-AM, and changes in intracellular
Ca2+ were measured after addition
of indicated stimuli, as described in EXPERIMENTAL
PROCEDURES. Arrows indicate time of addition of the
drugs. The following conditions are shown: 100 ng/ml ET-1
(A); cells pretreated with 100 µM
forskolin before stimulation with 100 ng/ml ET-1
(B); cells exposed to 100 ng/ml PMA
for 24 h before ET-1 (C); EGTA (5 mM) added before ET-1 (D); BAPTA-AM
(30 µM) added before ET-1 (E); 100 nM thapsigargin (Tg, F); treatment
with 100 nM thapsigargin for 8 h before addition of ET-1
(G). Data are representative of at
least 7 separate cell tracings obtained from 2 independent experiments
for each condition.
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Effect of H-7 and herbimycin A on sequential induction of expression
of ets-1 and collagenase I mRNA by ET-1.
There is growing evidence that ets-1
is involved in transcriptional control of matrix-degrading proteinases
such as collagenase I, stromelysin 1, and the urokinase-type
plasminogen activator. Inhibitors of
ets-1 mRNA expression in response to
ET-1 such as herbimycin A and H-7 (Fig.
11A)
also resulted in abrogation of expression of collagenase I mRNA (Fig.
11B). Interestingly, expression of
stromelysin mRNA was abundant even in serum-deprived SMC and only
modestly induced by ET-1 (data not shown).

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Fig. 11.
Sequential activation of ets-1 and
collagenase mRNA levels by ET-1 in VSMC: effects of H-7 and herbimycin
A (HA). Time course of regulation of
ets-1
(A) or collagenase I mRNA
(B) by ET-1 in the presence or
absence of either H-7 or herbimycin A. Abundance of indicated
transcripts was calculated by densitometry relative to that of GAPDH
mRNA, used as an internal control for uniformity of loading and
transfer.
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DISCUSSION |
The c-ets-1 protooncogene is believed
to transactivate the promoters of a number of cellular genes coding for
growth factors, proteins involved in the immune response, and
extracellular matrix-degrading proteinases (4). Because of its
relatively restricted pattern of expression during embryogenesis, in
which it is preferentially detected in lymphoid tissues, there has been
considerable attention focused on its possible role in T cell
maturation in the fetal thymus and T cell activation in adults (4).
However, c-ets-1 expression is also
seen in mesenchymal and endothelial cells of the developing embryo
(27). Its expression has also been noted in stromal cells and capillary
vessels at the margins of tumor invasion (30). Recently,
c-ets-1 expression was observed in periluminal arterial SMC following balloon injury (12). The pattern of
expression in human cancer fibroblasts and VSMC is consistent with
induction of expression mediated by extracellular stimuli, presumably
by growth factors or cytokines. Notably, there is scant information on
which factors may be involved in regulating ets-1 gene expression and of the
signal transduction pathways implicated. The transcriptional activity
of ets-1 is modulated in part through
a ras-mediated threonine
phosphorylation in its conserved amino-terminal domain, resulting in
superactivation of reporter systems driven by promoters containing
ets/AP-1 binding sites (31). However,
although the activity of ets-1 is
subject to regulation through
ras-mediated posttranslational
modifications of the protein, before this study it was not clear
whether the overall abundance of ets-1
was also subject to regulation by growth factors. In this study, we
describe that ets-1 fulfills the
characteristics of an early response gene in arterial SMC and that it
is subject to regulation by a PKC-mediated signal transduction pathway
activated by growth factors such as PDGF-BB and ET-1.
Treatment of VSMC with the protein synthesis inhibitor cycloheximide
resulted in clear superinduction of
ets-1 mRNA in response to both ET-1
and PDGF-BB. This indicates that neither of these growth factors
requires synthesis of an intermediary protein(s) to induce
ets-1 mRNA. The accumulation of
ets-1 mRNA in the presence of
cycloheximide alone may be due to either the abrogation of synthesis of
a repressor of ets-1 transcription or
of a factor accelerating its degradation. While this paper was in
preparation, Gilles et al. (8) reported that
ets-1 mRNA was also superinduced in
human fibroblasts in response to tumor necrosis factor-
. To our
knowledge, the categorization of ets-1
as an early response gene is novel and has significant implications.
Structural analysis of the human ets-1
promoter identified one binding site each for AP-1, AP-2, and
ets-1 itself (17). Functional studies
demonstrated that exogenous expression of
c-jun, AP-2, and
ets-1 were associated with activation
of the ets-1 promoter (17). The
evidence shown here that PDGF-BB and ET-1 induction of
ets-1 mRNA does not require new
protein synthesis indicates that generation of intermediary transcription factors such as ets-1,
c-jun, and AP-2 is not essential for
expression of the ets-1 gene.
Of the various growth factors tested, PDGF-BB and ET-1 were the most
powerful stimuli of ets-1 mRNA in
quiescent SMC. Interestingly, PDGF-BB and ET-1 interact with receptors
belonging to two distinct families (and couple to particular sets of
signaling intermediates) that converge on PKC through alternative
routes. After PDGF binds and activates its specific receptor tyrosine
kinase, it induces tyrosine phosphorylation of PLC-
. The stimulation
of PLC-
results in phosphatidylinositol 4,5-bisphosphate
(PIP2) hydrolysis, with consequent generation of inositol 1,4,5-trisphosphate and
diacylglycerol (DAG), leading to
[Ca2+]i
mobilization and PKC activation, respectively. On the other hand, ET-1,
originally isolated as a vasoconstrictor peptide, interacts with at
least two subtypes of membrane-anchored serpentine proteins belonging
to the family of G protein-coupled receptors. ET-1 stimulates PLC-
through G protein activation and PLC-
through autophosphorylation of
nonreceptor tyrosine kinases such as
pp60c-src
(14, 24). Indeed, PDGF-BB and ET-1 are known to evoke the expression of
c-fos and
c-jun mRNA levels through PKC
and/or protein tyrosine kinase activity in mesangial cells,
fibroblasts, and VSMC (19, 21, 23, 25, 29). There is increasing
evidence that AP-1 cooperates with
ets-1 in the transcriptional
stimulation of genes for matrix-degrading enzymes, which heightens the
significance of the coactivation of
c-ets-1,
c-jun, and
c-fos gene expression by PKC-mediated
pathways.
In contrast to the extensive investigations on the role of
ets transcription factors on the
function of various gene promoters, the signal transduction pathways
involved in the control of ets-1 gene
expression are poorly understood. We showed that H-7, a PKC antagonist,
inhibited ets-1 mRNA expression by
both PDGF-BB and ET-1. Because the action of H-7 is not entirely
specific to PKC, other lines of evidence were pursued to substantiate
signaling through this pathway in the control of
ets-1 mRNA abundance. The role of PKC
activation in this process was even more apparent after chronic PMA
pretreatment, which leads to PKC isozyme downregulation and
degradation, which was associated with almost complete abrogation of
PDGF-BB-, ET-1-, or acute PMA-stimulated
ets-1 gene expression. We observed
that inhibition of tyrosine kinase activity by herbimycin A, an
antibiotic that irreversibly inhibits the auto- and
transphosphorylation of
pp60c-src
and abolishes PLC-
tyrosine phosphorylation in VSMC (22, 26, 29),
strongly blocked ets-1 mRNA expression
by ET-1. The inhibition of tyrosine phosphorylation of PLC-
, with
consequent impairment in the hydrolysis of
PIP2 to
IP3 and DAG, may play an essential role in the activation of downstream components in the signal transduction for ets-1 gene expression
by ET-1. Herbimycin A had an effect of lesser magnitude on
PDGF-BB-induced ets-1 mRNA. These data
indicate that PDGF-BB-stimulated ets-1
gene expression might not be entirely dependent on PLC-
, since this
enzyme is known to be functionally inactivated in SMC at the range of
herbimycin A concentrations used, suggesting that PKC may be partially
activated by PDGF-BB through alternative routes involving other
components of glycerolipid metabolism, such as arachidonic acid (6). In summary, the acute stimulatory effects of phorbol esters, as well as
the inhibition by chronic PMA pretreatment and H-7, strongly point to a
key role for PKC activation in the induction of
ets-1 gene expression by both PDGF-BB
and ET-1 in VSMC.
Pharmacological treatment with forskolin activates adenylate cyclase,
and the resulting increase in cellular concentration of cAMP leads to
stimulation of PKA activity. Forskolin inhibited basal
ets-1 mRNA in SMC and almost
completely abrogated the phorbol ester, PDGF, and ET-1 induction of
ets-1 mRNA. cAMP regulation of gene
expression is chiefly considered to occur via CREB binding to target
regulatory elements in the promoter region of the target gene. The
human ets-1 promoter contains binding
sites for PEA3, AP-1, AP-2, and Sp1; however, CREB binding sites have
not been detected, suggesting that the impairment of
ets-1 gene expression may occur
through interference with more proximal signaling events (11, 17).
Activation of PKA is thought to lead to the inhibition of stimulated
PIP2 hydrolysis (1, 2) and
inhibition of
[Ca2+]i
released from the ER, probably via phosphorylation of
IP3 receptors (13). Under the
experimental conditions used here, we observed that forskolin partially
inhibited inositol phosphate generation by ET-1, although this did not
appear to reduce the magnitude of ET-1-induced intracellular
Ca2+ release. These findings
indicate that the cAMP/PKA-mediated inhibition of
ets-1 mRNA expression by ET-1 is not
likely to result from reduction of
IP3-mediated
Ca2+ release. Consistent with what
has been previously reported, the decrease in DAG production through
inhibition of PIP2 breakdown may
have resulted in partial inhibition of PKC activity and consequently in
inhibition of ets-1 gene expression.
Rothman et al. (21) observed that acute thapsigargin stimulation caused
a significant increase in a subset of
c-jun and c-fos mRNAs in pulmonary VSMC,
implicating a direct role of intracellular Ca2+ release from the ER in
transcriptional control of a set of early response genes, effects that
were independent of PKC activation (21). Furthermore, they showed that
the depletion of
[Ca2+]i
stores by chronic thapsigargin pretreatment blocked
egr-1 and fra-1 mRNA induction by thrombin, but
not by PDGF, whereas chelation of extracellular
Ca2+ with EGTA inhibited the
induction of a subset of c-jun and
c-fos mRNAs by both thrombin and PDGF.
In the present study, short-term treatment with thapsigargin itself did
not induce ets-1 mRNA in VSMC.
However, chelation of
[Ca2+]i
by BAPTA or depletion of ER Ca2+
stores by prolonged thapsigargin treatment inhibited
ets-1 mRNA expression by PDGF-BB,
ET-1, and PMA. These data suggest that the
ets-1 gene is regulated through a
signaling pathway distinct from that controlling
c-jun and
c-fos gene expression. They indicate that release of
[Ca2+]i
from ER stores is required for appropriate
ets-1 gene expression in response to
ET-1 and PDGF-BB. However, the lack of effect of acute treatment with
thapsigargin shows that, as opposed to the regulation of
c-fos and
c-jun in pulmonary SMC,
[Ca2+]i
release alone is not sufficient to induce
ets-1 mRNA. This is further supported
by the fact that, whereas downregulation of PKC by chronic PMA
treatment strongly inhibited ets-1
mRNA expression by ET-1, it did not have a significant effect on the [Ca2+]i
increase caused by ET-1. It is possible that the modest effects of
acute treatment with thapsigargin on
ets-1 mRNA may be due to the slow
elevation of intracellular Ca2+
elicited by this agent and that a more abrupt rise that more closely
mimics the pattern seen after exposure to ET-1 would have a more robust
effect. Alternatively, a theoretical basis for these results can be
proposed on the basis of recent evidence indicating that depletion of
Ca2+ from the lumen of the ER and
nuclear envelope with ionophores or thapsigargin rapidly and potently
inhibits passive diffusion and signal-mediated transport of proteins
into the nucleus (16). Intracellular
Ca2+ stored in the ER may be a
major component required for nuclear transport of signals generated
after PKC activation, although the precise mechanisms of how this may
occur are unclear.
As mentioned above, ets-1 is believed
to play a role in the transcriptional activation of matrix
metalloproteinases. Agents that interfered with
ets-1 gene expression in response to
ET-1 (i.e., H-7, herbimycin A) also impaired stimulation of collagenase I. In contrast, stromelysin mRNA was not induced by either PDGF-BB or
ET-1, irrespective of a brisk stimulation of
ets-1 gene expression. Although they
do not prove causality, these results are consistent with a role for
ets-1 in the regulation of collagenase
gene expression. Because the promoter regions of both the collagenase I
and stromelysin genes have ets-1
binding sites (PEA3 motifs), it is possible that stromelysin activation
requires other cofactors not present after ET-1 stimulation or that its
expression is only subject to ets-1 regulation in other cell types. The same pattern of collagenase I and
stromelysin gene regulation was also observed after treatment with
PDGF-BB (not shown).
In conclusion, the present studies demonstrate that
ets-1 functions as an early response
gene after mitogen stimulation. PKC activation and intracellular
Ca2+ stored in ER are pivotal
components of the signal transduction pathway for activation of
ets-1 gene expression by PDGF-BB and ET-1. In turn, ets-1 may play a key
role in VSMC migration or in arterial remodeling through control of
expression of matrix-degrading metalloproteinases.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-43802. J. A. Fagin is a recipient of an Established
Investigator Award of the American Heart Association and Bristol
Myers-Squibb.
 |
FOOTNOTES |
Address for reprint requests: J. A. Fagin, Division of Endocrinology
and Metabolism, Univ. of Cincinnati, PO Box 670547, Cincinnati, OH
45267-0547.
Received 14 April 1997; accepted in final form 6 November 1997.
 |
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