From the Department of Pharmacology and Toxicology
and § Department of Physiology, Biocenter Oulu,
University of Oulu, P. O. Box 5000, Oulu FIN-90014, Finland
Received for publication, June 6, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Terminally differentiated cardiac myocytes adapt
to mechanical and neurohumoral stress via morphological changes of
individual cells accompanied by reactivation of fetal pattern of gene
expression. Endothelin-1, a powerful paracrine mediator of myocyte
growth, induces similar changes in cultured cardiac myocytes as those seen in hypertrophied heart in vivo. By using rat B-type
natriuretic peptide promoter, we identified a novel ETS binding
sequence, on which nuclear protein binding is activated in
endothelin-1-treated cultured cardiac myocytes. This sequence binds
ETS-like gene-1 transcription factor and mediates endothelin-1-specific
activation of transcription, but not responses to increased calcium
signaling via L-type calcium channels, angiotensin II
treatment, or mechanical stretch of myocytes. Interestingly,
endothelin-1 activated signaling converges via p38 mitogen-activated
protein kinase-dependent mechanism on ETS binding site,
whereas this element inhibits extracellular signal-regulated kinase
activated transcription. In conclusion, given the fundamental role of
the interaction of mitogen-activated protein kinases and ETS factors in
regulation of eukaryotic cell differentiation, growth, and oncogenesis,
these results provide the unique evidence of a endothelin-1- and
mitogen-activated protein kinase-regulated ETS factor pathway for
cardiac myocytes.
Terminally differentiated cardiac myocytes, due to their inability
to divide, adapt to increased mechanical load and the activation of the
neurohumoral system by hypertrophy. Initiation of hypertrophic growth
is accompanied by a rapid and transient expression of immediate early
genes (e.g. c-jun, c-fos, and
Egr-1) followed by activation of a pattern of cardiac genes,
including atrial and B-type natriuretic peptide
(ANP1 and
BNP) and There are two distinct G-protein-coupled receptor (GPCR) subtypes for
ET-1, ETA and ETB receptors, both of which are
widely expressed in a variety of tissues, including myocardium (7). Activation of GPCRs catalyzes exchange of GTP for GDP within the heterotrimeric G-protein complex releasing activated G The mechanisms connecting ET-1-induced cytosolic signaling to nuclear
targets are poorly understood in cardiac myocytes. ET-1 has been
reported to activate nuclear factor- Chemicals--
Specific antibodies raised against transcription
factors of activator protein-1 (AP-1) complex (c-fos,
c-jun, jun-B, and jun-D), NFATc3,
NFATc4, ETS-like gene-1 (Elk-1), Ser-383-phosphorylated Elk-1,
ETS-1/-2 (DNA binding domain), Fli-1, GATA-4, NF Plasmids and Oligonucleotides--
All oligonucleotides were
from Sigma Chemical Co. except the NF Cell Culture and Transfection--
Neonatal rat ventricular
myocytes were prepared from 2- to 4-day-old Sprague-Dawley rats and
transfected with liposome-mediated transfection (20). The Animal Use
and Care Committee of the University of Oulu approved the experimental
design. Briefly, after preparation, cells were plated at the density of
2 × 105/cm2 on cell culture plates
(Falcon) or on collagen I-coated elastomere plates with flexible
bottoms (Bioflex, Flexcell Int.) for stretch experiments and cultured
overnight with Dulbecco's modification of Eagle's medium/Ham's F-12
medium (1:1) containing 10% of fetal bovine serum. Transfection of
cells (if designated) was performed on the second day in culture. Cells
were exposed to 3 µl of FuGENE 6 and 1.5 µg of DNA (1 µg of Luc,
0.5 µg of Northern Blot Analysis and Radioimmunoassay for BNP--
RNA was
isolated from cardiac myocytes by the guanidine thiocyanate-CsCl method
(21). Northern blots were hybridized with specific cDNA probes for
rat BNP, rat ANP, and rat ribosomal 18 S (20) labeled with
[32P]dCTP (Amersham Biosciences) with a T7 Quick Prime
kit (Amersham Biosciences). To measure the secretion of BNP, samples of
incubation medium were subjected to radioimmunoassay of BNP (20).
EMSA--
Nuclear extracts from cardiac myocytes (22) were
prepared and protein concentration from each sample was
colorimetrically determined (Bio-Rad Laboratories). Double-stranded
oligonucleotide probes were sticky end-labeled with
[32P]dCTP by Klenow enzyme (22). For each reaction
mixture (20), 6 µg of nuclear protein and 2 µg of poly(dI-dC) were
used in a buffer containing 10 mM HEPES (pH 7.9), 1 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol,
0.025% Nonidet P-40, 0.25 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml each leupeptin, pepstatin, and aprotinin.
Reaction mixtures were incubated with a labeled probe for 20 min
followed by gel-electrophoresis (250 V, for 1.5-1.75 h normally and
for supershift 2.5 h) on 5% polyacrylamide gel. Subsequently,
gels were dried and exposed in a PhosphorImager screen and analyzed
with ImageQuaNT (Amersham Biosciences). To confirm DNA sequence
specificity of the protein-DNA complex formation, competition
experiments with 10, 50, and 100 molar excesses of unlabeled
oligonucleotides with intact or mutated binding sites were performed.
In addition, DNA binding activity assays of Octamer-1 (Oct-1)
transcription factor were run in parallel to control the specificity of
the altered DNA binding activity (16). For competition and supershift
experiments appropriate competitor oligonucleotides or antibodies were
added to reaction mixture 20 min before labeled probe.
Immunoblot--
Western blot analysis was done using standard
reagents and protocol (16). Briefly, 20 µg of nuclear proteins was
used for SDS-PAGE, followed by Western blotting utilizing antibodies
against phospho-Elk-1 (Ser-383) and Elk-1 (binds both phosphorylated
and dephosphorylated Elk-1). Finally, blots were developed with goat anti-Rabbit IgG-HRP and enhanced luminescence reagents followed by
exposure of film for 1-3 min (Hyperfilm ECL, Amersham Biosciences).
Sarcomeric Organization--
Cardiac myocytes were grown on
collagen-coated glass coverslips and exposed to ET-1 (100 nM) for 24 h. Statistical Analysis--
Results are expressed as mean ± S.E. To determine the statistical significance between two groups and
for analysis of multiple groups, the Student's t test and
one-way analysis of variance followed by the least significant
difference post hoc test were used, respectively. Differences at
the 95% level were considered statistically significant.
ET-1 Activates BNP Gene Transcription via a Novel ETS Binding
Sequence--
Cultured neonatal ventricular myocytes serve as a useful
experimental model for the study of myocyte growth and the
intracellular mechanisms involved. Primary neonatal ventricular
myocytes (referred to hereafter as cardiac myocytes) used in this study
respond to a variety of stimuli by the activation of hypertrophic
growth program virtually identical to that of developing neonatal and the pathologic adult myocardium (5). ET-1 activated cardiac genes
associated with hypertrophic growth program, such as ANP and BNP (Fig.
1A), of which BNP synthesis
was augmented through an ETA receptor-dependent
mechanism (Fig. 1B) (23). In addition, cardiac myocytes
subjected to ET-1 develop hypertrophy, including activation of protein
synthesis (16, 20) and increased cell size accompanied by
reorganization of sarcomeres (Fig. 1C).
In the context of the rBNP promoter, previous studies have indicated
that the proximal 5'-flanking region of 535 bp is sufficient to confer
the induction by GPCR agonists (20, 24). The proximal ET-1 Activates Specific Complex Formation of Cardiac Nuclear
Proteins with EBS--
Previously, it has been demonstrated that
exposure of cardiac myocytes to hypertrophic agonists selectively
activate transcription factors followed by specific DNA binding
affinity and eventually activation of transcription (16, 26). To
analyze whether there is specific binding activity on EBS of rBNP
promoter by cardiac nuclear proteins, we utilized EMSA, and, when
designated, performed the protein-DNA binding reactions in the presence
a 100-molar excess of unlabeled competitor oligonucleotides (Fig.
3A). We found that three
specific shifts (complexes C1-3) were constantly formed, of which C1
and C2 migrated closely to each other separately from a faster
migrating complex C3. Competitor oligonucleotides with mutated
nucleotides from
In view of specific complex formation of cardiac nuclear proteins with
EBS of BNP promoter, we analyzed whether ET-1 treatment of cardiac
myocytes had any effect on the nuclear protein binding activity on EBS
(Fig. 4, A-D). Initially, a
rapid and transient increase in formation of complex C2 was detected at
5 and 15 min. Later, a sustained increase of EBS binding activity was
observed at 3 h for complexes C1 and C3, which remained elevated
at 24 h. In agreement with our previous studies (16), all the
nuclear extracts were tested in parallel for Oct-1 binding activity,
which was not altered by ET-1 suggesting a specific activation of EBS binding (data not shown). Taken together, ET-1 treatment of cardiac myocytes initiated a biphasic activation of protein binding on the EBS
of rBNP, and these specific complexes closely resembled complex
formation of nuclear proteins with a high affinity ETS binding
sequence.
Elk-1 Binds on EBS of rBNP Promoter--
Functional ETS factor
binding regulatory cis sequences have been characterized
extensively in both viral and eukaryotic genes, but there are
exceptionally few reports of the cardiac genes regulated by ETS factors
(28). Gupta et al. (29) have reported a repressor role for
3'-flanking EBS resulting in myocyte-restricted expression of
Given the cumulative evidence demonstrating that Elk-1 transcription
factor is regulated via site-specific phosphorylation (32), we further
investigated activation of Elk-1 by using Western blot analysis. The
site-specific phosphorylation (Ser-383) of Elk-1 transiently increased
at 5 and 15 min in cardiac myocytes treated with ET-1 (Fig.
5B). Therefore, Elk-1 phosphorylation is temporally related
to increased formation of complex C2 in EMSA (Fig. 4B). In
addition, antibodies raised against NFATc3, NFATc4, GATA-4, STAT-1 and
-3 and competitor oligonucleotides of AP-1, GATA-4 binding sequences
( ET-1 Activates rBNP Transcription via EBS by p38
MAPK-dependent Mechanism--
Hypertrophic stimuli,
including GPCR agonists (e.g. ET-1, phenylephrine, and Ang
II) and mechanical stress activate MAPKs in cardiac myocytes (14, 16,
33). Elk-1 transcription factor is a known downstream target for MAPKs
(34, 35), and thus we investigated their potential role in activation
rBNP transcription via EBS by using expression plasmids for
each MAPK pathway. A minimal amount of expression plasmids (0.05 µg)
was selected to prevent quenching and to confirm that the effects of
overexpression were specific for each MAPK cascade. Dominant negative
(dn) kinases of MEK1 (upstream of ERK), MKK6 (upstream of p38), and
c-Jun N-terminal kinase-1 (JNK) were utilized to inhibit specifically
each pathway. Interestingly, only the inhibition of p38
MAPK-dependent mechanism attenuated the activation of
rBNP transcription by ET-1 (Fig. 6A). Vice versa, the
activation of p38 MAPK resulted in EBS-dependent activation
of rBNP promoter activity (Fig. 6B). Contrastingly, activation of ERK signaling by forced expression of MEK1 activated rBNP transcription only when signaling via EBS was blocked
by mutation (Fig. 6B). Furthermore, pharmacological blockade
of p38 MAPK pathway by p38 MAPK inhibitor (SB203580, 20 µM) prevented ET-1 (100 nM, 15 min)-induced
Ser-383 phosphorylation of Elk-1 (data not shown).
In addition to MAPK cascades, ET-1 is a known activator of
Ca2+-dependent signaling such as CaMK and
calcineurin in cardiac myocytes (13). Therefore, we further examined
whether activation of Ca2+-dependent pathways
induced rBNP transcription similarly via the p38 MAPK- and
EBS-dependent mechanism as ET-1. Cells were exposed to
dihydropyridine agonist Bay K8644 to activate Ca2+ influx
via L-type Ca2+ channels (36). Bay K8644
induced rBNP transcription independently of EBS, and the
induction was equally inhibited by dominant negative expression
plasmids of each MAPK cascade (Fig. 6C). Similar results were obtained with physiological activation of L-type
Ca2+ channels using The founding member of the ETS family in mammalians, ETS-1, was
originally discovered due to homology to v-ets, an oncogene of avian E26 ("E-Twenty-Six") retrovirus genome (37). ETS factors share a winged helix-turn-helix domain for DNA binding, and they bind
to 5'-GGA(T/A)-3' ETS binding sequence (EBS) (27, 38). ETS
factors have been implicated in transforming and tumor-associated products as well as cell cycle and apoptosis regulator genes, and they
regulate hematopoiesis and the development of central nervous system,
bone and cartilage, mammary glands, and vasculature (28, 39).
Therefore, the identification of target genes for ETS signaling has
emerged to a major area of research interest.
Although there are multiple ETS factors in the heart (40), their
functional role has not been revealed. Activation of c-fos promoter via Elk-1 together with SRF composes a regulatory mechanism of
c-fos gene present in most eukaryotic cell types, including cardiac myocytes (30, 31). In addition, ETS-domain transcription factors act through EBS containing no adjacent SREs, likely in complex
with alternative cofactors, but only a single report is available of
such a regulatory EBS acting on a gene expressed in myocytes
( Importantly, treatment of myocytes with ET-1 activated specific complex
formation between nuclear proteins and EBS of BNP. Similar activation
of binding affinity on rBNP gene has been reported of GATA-4
transcription factor (16). Moreover, it is evident that activation of
rBNP gene by forced expression of p38 MAPK alone requires
both intact EBS and GATA elements (16) at the proximal promoter region.
However, ERK is also capable of phosphorylating GATA-4 in a manner
similar to p38 MAPK (16), but it appears that activation of rBNP
promoter by the ERK pathway is inhibited by signaling via EBS
(schematic presentation in Fig. 7). This model of nuclear regulation may target parallel MAPK pathways in a
stimulus-dependent manner. Our hypothesis is supported by previous studies indicating that activation of rat BNP promoter during
myocyte stretch appears to require intact GATA binding sequence at
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-myosin heavy chain
(
-MHC), whereas relative expression of a subset of genes
is simultaneously decreased (i.e.
-myosin heavy
chain,
-MHC) (1, 2). Natriuretic peptides, released into circulation by cardiac myocytes, defend the cardiovascular system
against increased hemodynamic load and decrease blood pressure by
increasing salt and water excretion and by promoting vasodilation (3).
However, when both mechanical and neurohumoral stress of myocytes are
sustained, adaptive mechanisms fail leading to maladaptation and,
eventually, cardiac dysfunction (i.e. congestive heart
failure) (4). Emerging evidence suggests that a number of humoral
factors, such as angiotensin II (Ang II) and endothelin-1 (ET-1),
participate in this adaptive process and modify the growth of cardiac
myocytes (5). Production of ET-1 is increased in humans with cardiac
hypertrophy and congestive heart failure, and ET-1 plasma
concentrations correlate with the clinical severity of the cardiac
failure (6).
-GTP and G
subunits, each of which has regulatory functions (8). Binding of ET-1 to its receptors induces
Gq/11-protein-dependent stimulation of
phospholipase C, leading to formation of inositol 1,4,5-triphosphate and diacylglycerol (9), which activate the release of calcium from
intracellular stores and protein kinase C (10), respectively. Altered
calcium handling of myocyte, a feature of human heart failure
(11), may result in activation of
calcium/calmodulin-dependent kinase (CaMK) and phosphatase
(i.e. calcineurin) pathways (12), both of which are
implicated in endothelin-1-induced signaling cascades (13). In
addition, signaling pathways downstream of ET receptors involve small
G-proteins (Ras, RhoA, and Rac) accompanied by activation of
mitogen-activated protein kinases (MAPKs) (14).
B (NF
B) transcription factor
(15) and, via MAPK-mediated phosphorylation, GATA-4 transcription factor (16). Physical and functional interactions of GATA-4 with its
cofactors, nuclear factor of activated T lymphocytes (NFAT) and serum
response factor (SRF), are induced in ET-1-treated cardiac myocytes
(17, 18). However, inhibitory strategies targeted against either GATA-4
protein synthesis or DNA binding affinity have produced
different results in ET-1-induced hypertrophic growth of cardiac
myocytes, suggesting an increasing complexity of
ET-1-dependent pathways (19, 20). To elucidate the nuclear mechanisms of ET-1-activated cell signaling, we studied rat
BNP (rBNP) as a cardiac myocyte-specific target
gene for ET-1 and identified a novel signaling mechanism initiating
from cell membrane ETA receptor and leading to p38 MAPK-
and ETS factor-dependent induction of the rBNP gene.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (p50, p65),
Nkx-2.5, and SRF were from Santa Cruz Biotechnology (San Diego, CA).
Specific antibodies against signal transducers and activators of
transcription-1 and -3 (STAT-1 and -3) were purchased from Upstate
Biotechnology (Lake Placid, NY). FuGENE 6 transfection reagent was from
Roche Molecular Biochemicals (Indianapolis, IN). ET-1, Ang II, and Bay
K8644 were obtained from Sigma Chemical Co. (St. Louis, MO), BQ610 was
from Peninsula Laboratories Inc. (San Carlos, CA), and bosentan was a
kind gift from Dr. M. Clozel (Actelion Pharmaceuticals Ltd., Basel,
Switzerland). Anti-rabbit IgG antibody linked to horseradish peroxidase
(Anti-rabbit IgG-HRP, New England BioLabs Ltd., Hertfordshire, UK) and
ECL+Plus reagents (Amersham Biosciences, Buckinghamshire, UK) were used
for Western blot detection.
B and shift-inducible element
electrophoretic mobility shift assay (EMSA) probes were from Santa Cruz
Biotechnology. Plasmid for Rous sarcoma virus-promoter linked to
-galactosidase gene (RSV-
gal) and pGL3-Basic
plasmid-expressing luciferase gene were purchased from
Clontech (Palo Alto, CA). Mutations (QuikChange site-directed mutagenesis, Stratagene, La Jolla, CA) to the
534 bp
5'-flanking region of rBNP promoter-driven pGL3-Basic plasmid (
534BNP-luc) (20) were introduced using oligonucleotides (coding strand, mutated nucleotides are in boldface) of
5'-GCTACCAGAGTGCCCAGCCTCCGTGCAGCCCGGCCC-3', 5'-CTGGAAGTGTTTTTGACAGTTCACCCCATAAAGCCCC-3' and
5'-GGCAGGAATGTGTCTTGCAAATCAGATGCAACCCCACCCCTAC-3', for ETS binding sequence at
498 bp, AP-1 binding sequence at
373 bp
and tandem GATA binding sequence at
90/
81 bp of rBNP promoter
(GenBankTM accession number M60266), respectively.
All mutations were confirmed by nucleotide sequencing (ABI-Prism 310, PerkinElmer Life Sciences, Foster City, CA). Plasmids encoding wild
type and dominant negative mutant of MEK1 (pCMV-MEK1, pCMV-MEK1(K97M)) were a kind gift from Dr K. L. Guan (University of Michigan, Ann Arbor, MI), a dominant negative MKK6 (pCMV-MKK6AL) from Dr. J. R. Woodgett (University of Toronto, Canada), and plasmids expressing of
p38
MAPK (pCMV-FLAG-p38
) and wild type and dominant negative mutant c-Jun N-terminal kinase-1 (pCDNA-FLAG-JNK1,
pCDNA-FLAG- JNK1(APF)) from Dr. R. Davis (University of Massachusetts).
-gal plasmids) per milliliter of complete serum-free
medium for 6 h and cultured thereafter in complete serum-free
medium. Cells on Bioflex plates were exposed to cyclic mechanical
stretch. Frequency of cyclic stretch was 0.5 Hz with pulsation of
10-25% elongation of cells for 24 h. Cells were stretched by
applying a cyclic vacuum suction under Bioflex plates by
computer-controlled equipment (FX-3000, Flexcell Int.). In
co-transfection experiments, cells were grown on 24-well plates and
transfected with 0.45 µg of intact
534BNP-luc or with
534BNP-luc
containing mutation of EBS and 0.05 µg of expression plasmid (see
"Plasmids and Oligonucleotides") of dnMEK1, dnMKK6, dnJNK1, MEK1,
p38
, JNK1, or pMT2 plasmids with 0.25 µg of RSV-
gal and 1.5 µl of FuGENE 6 per milliliter. Empty control plasmid pMT2 was used to
equalize the amount of transfected DNA between experimental groups. All
experiments were started subsequently on the third day, and cells were
harvested according to the experiment during the third or fourth day in
culture. Reporter gene activities were measured by using luciferase and
-galactosidase (to correct transfection efficiency) assays (Promega)
with luminometer. Luciferase activity levels were 9.2 ± 1.0 (average ± S.E.)-fold higher than those of
-galactosidase
activity. Both luciferase and
-galactosidase levels were at the
linear range of the assay measurement scale.
-Actin filaments of fixed myocytes
were labeled with Alexa FluorTM 488 Phalloidin (Molecular
Probes Inc., Eugene, OR) (20). Representative images were taken with
laser confocal microscope (LSM 510, Zeiss, Jena, Germany).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ET-1 induces hypertrophic phenotype of
cardiac myocytes. A, in cardiac myocytes treated
with ET-1 (100 nM), ANP and BNP mRNA levels increased.
After 24 h of ET-1 treatment, total RNA was extracted and
subjected to Northern blot analysis. Northern blots were hybridized
with specific cDNA probes for rBNP, rANP, and rat ribosomal 18 S
labeled with [32P]dCTP. ANP and BNP mRNA levels were
measured and corrected with 18 S mRNA levels. For each
bar, average ± S.E. values of six separate experiments
are shown. B, ET-1 activates BNP secretion via
ETA receptor. Cardiac myocytes were treated with ET-1 alone
(100 nM, 24 h) or with specific ETA
receptor antagonist (BQ610, 10 µM) or mixed
ETA/B receptor antagonist (bosentan, 1 µM).
After incubation, the amount of immunoreactive BNP in culture medium
was measured with radioimmunoassay. ET-1 increased immunoreactive BNP
concentration, which was equally abolished by BQ610 or bosentan,
suggesting an ETA receptor-mediated mechanism. For each
bar, average ± S.E. values of four separate
experiments are shown. C, activation of sarcomeric protein
organization of cardiac myocytes by ET-1. Cardiac myocytes grown on
collagen-coated glass coverslips were incubated with or without ET-1
(100 nM, 24 h). After fixation of cells, -actin
filaments were labeled with Alexa Fluor 488 phalloidin. Representative
images of five separate experiments were taken with laser confocal
microscope (LSM 510). *, p < 0.05 compared with
untreated control cells; #, p < 0.05 compared with
ET-1-treated cells.
534 bp of BNP
promoter contain multiple potential cis-acting elements (25) such as
GATA elements. Proximal GATA elements participate in the activation of
rBNP gene transcription by
-adrenergic agonist
phenylephrine (24) but not by ET-1 (20). Therefore, we utilized a
computer-based search for novel cis-acting elements of the rat BNP gene
and identified an ETS binding sequence (EBS) and AP-1 binding sequence
at
498 and
373 bp of the rBNP promoter, respectively. To
characterize the role of these elements, we introduced site-directed
mutations to EBS and AP-1 binding sequences and to
90/
81-bp tandem
GATA elements of the rBNP promoter (Fig. 2A). Endothelin-1 activated
proximal
534-bp rBNP promoter dose-dependently via
ETA receptor, and the induction was inhibited by mutation of EBS (Fig. 2, B and C). The combination of
mutations of AP-1 and/or GATA elements with the mutation of EBS did not
further decrease the inhibition seen with rBNP promoter containing
mutated EBS (data not shown). Importantly, the induction of
rBNP transcription in myocytes by mechanical stretch or Ang
II was unaffected by the mutation of EBS (Fig. 2D). To
investigate this ET-1 responsive element further, we studied the
complex formation of cardiac nuclear proteins with EBS of rBNP
promoter.
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Fig. 2.
Endothelin-1-specific activation of rBNP
promoter via novel ETS binding sequence (EBS). A,
schematic presentation of the binding sites of 534BNP-luc construct
with the mutated nucleotides of EBS, AP-1, and GATA motifs.
B, dose-dependent activation of rBNP promoter
activity via ETA receptor by ET-1. Cardiac myocytes were
transfected simultaneously with plasmids of
534BNP-luc and RSV-
gal
by using cationic liposome delivery. Transfected cells were treated
with increasing concentrations of ET-1 (0-100 nM) alone or
in the presence of specific ETA receptor blocker BQ610 (10 µM). After 24 h, cells were lysed and lysates were
assayed for luciferase and
-galactosidase activity to correct
transfection efficiency. *, p < 0.05 compared with
untreated control cells; #, p < 0.05 compared with 100 nM ET-1-treated cells. C, the effect of
site-directed mutations on the induction of rat BNP promoter by ET-1.
Cardiac myocytes were transfected with intact plasmid of
534BNP-luc
or with
534BNP-luc containing site-directed mutations (EBS (
498
mut), AP-1 (
373 mut), or GATA (
90/
81 mut)) and RSV-
gal by
using cationic liposome delivery. Transfected cells were subjected to
ET-1 (100 nM, 24 h), and subsequently cell lysates
were assayed for luciferase and
-galactosidase activity to correct
transfection efficiency. *, p < 0.05 compared with
induction of
534BNP-luc. D, the effect of EBS mutation on
534-BNP promoter activation by mechanical stretch and Ang II. Cardiac
myocytes were transfected with intact plasmid of
534BNP-luc or
534BNP-luc with site-directed mutation of EBS and RSV-
gal by using
cationic liposome delivery. Transfected cells were subjected to
cyclical stretch (frequency of 0.5 Hz with elongation between 10 and
25%) or Ang II (1 µM) for 24 h and subsequently
assayed for luciferase corrected with
-galactosidase activity. Data
are shown as -fold induction compared with control cells (transfected
with same luciferase construct). For each bar, average ± S.E. values of five (B), nine (C), or ten
(D) separate experiments are shown.
497 bp (mut 2) to
491 bp (mut 8) failed to compete
against intact EBS and were found essential for formation of complexes
C1-3 (i.e. 5'-cCCGGAAGtg-3'), suggesting similarity of
binding proteins in these complexes. Interestingly, a labeled high
affinity ETS binding sequence (SC1) (27) formed complexes similar to
C1-3 (Fig. 3A). The core sequence for ETS factor binding of
SC1 (Table I) is similar to EBS of rBNP
promoter. When SC1 oligonucleotide was used as a unlabeled competitor
against nuclear protein binding on EBS of rBNP promoter, SC1
dose-dependently inhibited protein binding on EBS, and the
competition was abolished by single nucleotide mutation of SC1
(i.e. 5'-CCGaAAGTG-3') (Fig. 3B). An NF
B
binding oligonucleotide (Table I), as an another potential GGA sequence
binding element, was not able to compete against binding on EBS of the
rBNP promoter (Fig. 3B). Moreover, with EBS of rBNP, nuclear
proteins extracted from adult rat heart (left ventricle) formed
identical complexes C1-3 in EMSA (data not shown), suggesting that ETS
factors binding on EBS of rBNP are present in adult heart.
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Fig. 3.
Characterization of 498 BNP ETS binding
sequence (EBS). A, competition analysis of ETS binding
sequence of rat BNP promoter and comparison with SC1 probe.
B, competition analysis for nuclear protein binding on ETS
binding sequence of rBNP promoter with NF
B binding site and SC1
oligonucleotides. EMSA using double-stranded oligonucleotide
corresponding to ETS binding sequence at
498 bp of rBNP promoter
((
498) BNP) and oligonucleotide corresponding to a high
affinity ETS binding site (SC1) as a radioactively labeled
probes was carried out with 100-molar excess of unlabeled competitor
oligonucleotides (for sequence see Table I). For the binding reaction,
6 µg of a mixed nuclear protein extract from untreated and ET-1 (100 nM, 15 min and 24 h)-stimulated cardiac myocytes was
used. Binding reactions with EMSA were repeated separately four times
from different nuclear extracts, and the three specific complexes
invariably formed are indicated as C1, C2, and
C3.
Comparison of oligonucleotides used in EMSA
498 bp are indicated by
boxes, the point mutations are in boldface, and
5'-overhangs are italic.
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Fig. 4.
Activation of nuclear protein binding on EBS
during ET-1 treatment. A, representative EMSA of C1-3
complex formation in ET-1-stimulated cardiac myocytes and the kinetics
of C1 complex (B), C2 complex (C), and C3 complex
formation (D) in EMSA using double-stranded oligonucleotide
corresponding to ETS binding sequence of rBNP promoter (( 498)
BNP) as a radioactively labeled probe was carried out with nuclear
proteins of cardiac myocytes treated with 100 nM of ET-1.
For binding reaction, 6 µg of nuclear protein extract of cardiac
myocytes was used. The three specific complexes are indicated as
C1, C2, and C3. For each bar,
average ± S.E. values of nine separate experiments for
B and D or of 13-14 for C are shown.
*, p < 0.05 and **, p < 0.01 compared
with untreated control cells.
-MHC gene, whereas other studies available have described an interaction of serum response element (SRE) with an EBS in the
activation of c-fos gene in cardiac myocytes (30, 31). EBS
of rBNP differs significantly from those reported previously: (i)
2.2
kb of the 5'-flanking region of rat BNP gene contains no potential SRE
for TCF (ternary complex factor)-SRF synergy (18) and (ii) EBS
functions as an activator element of rBNP transcription
exclusively by ET-1. To identify the transcription factors binding on
EBS, we used specific antibodies added to binding reaction prior
radioactively labeled EBS and subsequent EMSA. We utilized antibodies
for NF
B (p50 and p65), AP-1 complex (c-fos, c-jun, jun-B, and jun-D), Nkx-2.5,
SRF, ETS-1/-2 (DNA binding domain), Fli-1 (a member of ETS
transcription factor family), and activated Ser-383-phosphorylated
Elk-1 (Phospho-Elk-1). We found that ET-1-activated C2 complex was
supershifted by antibody raised against Phospho-Elk-1 (Fig.
5A). In contrast, none of
these antibodies had any effect on GATA-4 binding activity on the
90/
81-bp rBNP GATA binding site in EMSA run in parallel (data not
shown).
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Fig. 5.
Analysis of EBS binding proteins.
A, Elk-1 binds on EBS of rBNP and is a major protein in C2
complex. EMSA using double-stranded oligonucleotide corresponding to
EBS of rBNP (( 498) BNP) as a radioactively labeled probe
was used with 6 µg of nuclear proteins from cardiac myocytes treated
with ET-1 (100 nM, 15 min). For supershift reaction, 2.5 µl of specific antibodies raised against NF
B (p50 and
p65), the members of AP-1 complex, Nkx-2.5, SRF, ETS-1/-2
(DNA binding domain), Fli-1, or Ser-383-phosphorylated Elk-1 (P-Elk-1)
were added to the binding reaction. Supershifted C2 complex is
indicated (SS). B, ET-1 increases phosphorylation
of Elk-1. Nuclear proteins from control and ET-1-treated (100 nM) cardiac myocytes were subjected to SDS-PAGE. An
antibody raised against Ser-383-phosphorylated Elk-1 was used to reveal
the amount of activated Elk-1 protein (p-Elk-1) and total amount of
Elk-1 (Elk-1) was quantified by immunoblotting filters with another
antibody raised against Elk-1 that binds both phosphorylated and
dephosphorylated Elk-1 proteins. A representative Western blot of four
to five separate experiments for each time point is shown.
373 bp and
90 bp/
81 bp of rBNP) and Nkx-2.5 binding element (at
240 bp of rat ANP gene) or shift-inducible element failed
to have any effect on complex formation of cardiac nuclear proteins
with EBS (data not shown). In conclusion, ET-1 induces phosphorylation
and binding of Elk-1 on EBS of rBNP (complex C2) and likely activates
additional ETS related proteins whose identity we were unable to
identify by using specific antibodies against ETS related or other
transcription factors.
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[in a new window]
Fig. 6.
The role of MAPKs and
calcium-dependent pathways in the activation of rBNP
promoter. A, effect of dominant negative (dn) mutants
of MEK1, MKK6, and JNK1 on induction of BNP promoter by ET-1.
B, effect of mutated EBS on activation rat BNP promoter by
forced expression of MAPKs. C, effects of dn mutants of
MEK1, MKK6, and JNK1 and mutated EBS on activation of BNP promoter by
L-type Ca2+ channel agonist Bay K8644. Cardiac
myocytes were grown on 24-well Falcon plates, and each well was
transfected with 0.45 µg of intact 534BNP-luc or with
534BNP-luc
containing site-directed mutation of EBS and 0.05 µg of expression
plasmid of dnMEK1, dnMKK6, dnJNK1, MEK1, p38
, JNK1, or pMT2 (empty
control plasmid) with 0.25 µg of RSV-
gal and 1.5 µl of FuGENE 6. When indicated, transfected cells were subjected to treatment with ET-1
(100 nM) or Bay K8644 (1 µM) for 24 h.
Cell lysates were assayed for luciferase activity corrected with
-galactosidase activity. For each bar, average ± S.E. values of six separate experiments are shown. ***,
p < 0.001 compared with control cells transfected with
pMT2 plasmid.
1-adrenergic receptor
stimulation by isoproterenol (data not shown). Conclusively, these
results suggest that ET-1 activates rBNP gene transcription
via EBS in a p38 MAPK-dependent mechanism, whereas
induction of BNP gene by robust activation of
Ca2+-dependent signaling likely requires
activity of all three MAPK cascades in parallel. In contrast, intact
EBS of rBNP promoter inhibits the activation of transcription by the
ERK pathway alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MHC) (29). Yet, multiple genes activated during hypertrophic growth of cardiac myocytes contain potential EBS in their
5'-flanking regulatory region, such as genes for
-MHC and ANP (29).
In the present study, we identified a novel EBS at
498 bp of rBNP
promoter, constituting the rBNP gene as a first cardiac
target for ETS signaling that is activated during hypertrophic growth.
90/
81 bp (25) but not EBS at
498 bp of rBNP. Taken together with
the fact that ETS factors have been shown to regulate tissue-restricted
gene expression by utilizing both gene activation and repression
mechanisms (41, 42), this study provides unique evidence for the role
of ETS transcription factors as a gatekeeper of MAPK-mediated cardiac
gene activation.
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[in a new window]
Fig. 7.
Schematic presentation of ET-1-activated
signaling pathways converging on rBNP promoter. ET-1 activates
rBNP gene transcription in a MKK6/p38
MAPK-dependent manner via ETS binding sequence. Both p38
MAPK and MEK1/ERK phosphorylate GATA-4 transcription factor, whereas
intact ETS binding sequence inhibits induction of rBNP gene
transcription via the ERK pathway, leading exclusively to a p38
MAPK-dependent mechanism.
Elk-1, identified in protein complex binding on EBS of rBNP, is directly regulated via site-specific phosphorylation by MAPK pathways (34, 35) and conversely inactivated by dephosphorylation by phosphatases, including calcineurin (43). Originally, studies with T cells demonstrated that calcineurin dephosphorylates NFAT leading to nuclear translocation (44), but Molkentin et al. (45) described a role for the calcineurin- and NFAT-dependent signaling pathway also in myocyte hypertrophy. In the present study, robust activation of calcium signaling via L-type calcium channels resulted in the activation of BNP promoter independently of EBS, and all three MAPK cascades were equally required. Therefore, increased calcium signaling, mechanical stretch, and Ang II activate rBNP promoter via mechanisms that appear to uncouple the requirement of EBS of BNP gene. Calcineurin has been shown to direct signaling through JNK and ERK cascades (46, 47) and to inactivate signaling via Elk-1 through dephosphorylation (32). In addition, accumulation of intracellular calcium may lead to autoinhibition of DNA binding activity of ETS factors via site-specific CaMK-mediated phosphorylation (48). In conclusion, recent studies (49, 50) combined with our results suggest an intimate relationship between MAPKs and Ca2+-dependent pathways in the development of cardiac hypertrophy.
It is noteworthy that pathophysiological effects of ET-1 extend beyond myocardium to the regulation of proliferation (51) and contraction (52) of vascular smooth muscle cells, and the most notable effects are seen in coronary arteries (53). Therefore, it is intriguing to speculate that the ET-1-activated nuclear pathway described in the present study may not be limited to the regulation of cardiac gene expression but might extend to involve additional ETS factors expressed in vasculature, such as ETS-1, the expression of which is activated by ET-1 in cultured vascular smooth muscle cells (54) and in human endothelial cells during tumor angiogenesis (55). Interestingly, ET-1 increases vascular endothelial growth factor production in ovarian cancer cells via ETA receptor (56), whereas ETS factors activate genes regulating angiogenesis, such as vascular endothelial growth factor receptor R1 (57) and -2 (58). Furthermore, ET-1 is an endogenously produced growth factor implicated in tumor cell proliferation (59), and ETA receptor antagonism may prove useful treatment strategy in malignancies where this protein is overexpressed (60). Indeed, the ET-1-activated and MAPK-regulated ETS factor signaling provides an attractive target for the ongoing study of cellular growth, proliferation, and oncogenesis.
In summary, our results demonstrate that ET-1 activates rBNP
gene via mechanisms distinct from those of increased calcium influx via
L-type calcium channels, Ang II, or mechanical stretch of
myocytes. Importantly, ET-1 induced binding of ETS factors (Elk-1) on
EBS of rBNP promoter, and this element was required for the activation
of transcription by ET-1. The p38 MAPK-dependent mechanism
mediated activation of rBNP transcription via EBS, whereas activation
of rBNP promoter via the MEK1/ERK pathway was inhibited by intact EBS.
In conclusion, given the fundamental role of the interaction of MAPKs
and ETS factors in the regulation of eukaryotic cells, these results
provide the unique evidence of an ET-1-specific and MAPK-regulated ETS
factor pathway for cardiac myocytes.
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FOOTNOTES |
---|
* This work was supported by grants from the Academy of Finland (to H. R.), the AstraZeneca Research Foundation (to S. P.), the Sigrid Juselius Foundation (to H. R.), the Aarne Koskelo Foundation (to R. K., T. M.-P., and S. P.), the Ida Montin Foundation (to R. K. and S. P.), the Finnish Foundation for Cardiovascular Research (to H. R., S. P., and R. K.), the Research Foundation of Orion Co. (to R. K.), and the Finnish Cultural Foundation (to S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 358-8-537-5236; Fax: 358-8-537-5247; E-mail: heikki.ruskoaho@oulu.fi.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M205616200
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ABBREVIATIONS |
---|
The abbreviations used are:
ANP, atrial
natriuretic peptide;
BNP, B-type natriuretic peptide;
-MHC,
-myosin heavy chain;
Ang II, angiotensin II;
ET-1, endothelin-1;
GPCR, G-protein-coupled receptor;
CaMK, calcium/calmodulin-dependent kinase;
MAPK, mitogen-activated protein kinase;
SRE, serum response element;
SRF, serum response factor;
STAT, signal transducers and activators of
transcription;
Oct-1, octamer-1;
EMSA, electrophoretic mobility shift
assay;
EBS, ETS binding sequence;
NFAT, nuclear factor of activated T
lymphocyte;
AP-1, activator protein-1;
HRP, horseradish peroxidase;
CMV, cytomegalovirus;
ERK, extracellular signal-regulated kinase;
MEK1, MAPK/ERK kinase 1;
dn, dominant negative;
JNK, c-Jun
NH2-terminal kinase.
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