From the Jean Mayer USDA Human Nutrition Research
Center on Aging and ¶ Pulmonary and Critical Care Division,
Department of Medicine, Tufts University, Boston, Massachusetts 02111 and the ** Department of Developmental and Molecular Biology,
Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, November 14, 2002
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ABSTRACT |
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Hepatocyte growth factor (HGF) is released in
response to myocardial infarction and may play a role in regulating
cardiac remodeling. Recently, HGF was found to inhibit the apoptosis of cardiac muscle cells. Because GATA-4 can induce cell survival, the
effects of HGF on GATA-4 activity were investigated. Treatment of HL-1
cells or primary adult rat cardiac myocytes with HGF, at concentrations
that can be detected in the human serum after myocardial infarction,
rapidly enhances GATA-4 DNA-binding activity. The enhanced DNA-binding
activity is associated with the phosphorylation of GATA-4. HGF-induced
phosphorylation and activation of GATA-4 is abolished by MEK inhibitors
or the mutation of the ERK phosphorylation site (S105A), suggesting
that HGF activates GATA-4 via MEK-ERK pathway-dependent
phosphorylation. HGF enhances the expression of anti-apoptotic
Bcl-xL, and this is blocked by dominant negative mutants of MEK or GATA-4. Forced expression of wild-type GATA-4, but
not the GATA-4 mutant (S105A) increases the expression of Bcl-xL. Furthermore, expression of the GATA-4 mutant
(S105A) suppresses HGF-mediated protection of cells against
daunorubicin-induced apoptosis. These results demonstrate that HGF
protects cardiac muscle cells against apoptosis via a signaling pathway
involving MEK/ERK-dependent phosphorylation of
GATA-4.
The hepatocyte growth factor
(HGF)1 is a heterodimeric
protein composed of a 69-kDa Earlier findings that HGF expression is increased after liver injury
induced by CCl4 (9) suggest that HGF may be an oxidative stress-inducible factor. Similarly, in a rat model of myocardial ischemia and reperfusion, HGF expression was found to be enhanced (10).
Furthermore, human studies have shown that serum HGF levels are
elevated 25-50-fold after acute myocardial infarction (11, 12). HGF
has been postulated to serve as an endogenously produced cardioprotective factor (13). Recent studies supported this hypothesis
by demonstrating that gene transfection (14) or injection (15) of HGF
attenuated myocardial ischemia-reperfusion injury in rats. The
mechanism of HGF-mediated protection is at least, in part, because of
its actions on cardiac myocytes, as HGF has been shown to attenuate
oxidative stress-induced death of neonatal rat ventricular myocytes
(15), adult rat ventricular myocytes (16, 17), and HL-1 adult mouse
cardiac muscle cells (16). Furthermore, HGF was found to augment the
expression of antiapoptotic Bcl-xL both in vivo
and in vitro (15).
GATA-4 is a member of the GATA family of zinc finger transcription
factors, which plays important roles in transducing nuclear events that
modulate cell lineage differentiation during development. Six GATA
family members have been identified and shown to alter transcription of
target genes via binding to the consensus 5'-WGATAR-3' sequence. Three
members of this family, GATA-4/5/6, are expressed in the heart.
Functionally relevant GATA-binding sites have been identified in
numerous cardiac transcriptional regulatory regions (18, 19).
In addition to regulating differentiation, there is increasing evidence
that GATA factors also control cell survival. Weiss and Orkin (20)
reported that the GATA-1-deficient erythroid precursors undergo
apoptosis. In erythroleukemia cells, the induction of apoptosis by
estrogen was dependent on the inhibition of GATA-1 (21, 22). The
bcl-x gene has two GATA consensus motifs in the 5' promoter
region (23), and GATA-1 induces the expression of the antiapoptotic
protein Bcl-xL (24). GATA-1 also regulates the expression
of Bcl-2 (25). GATA elements are found in the promoters of other genes
involved in antiapoptotic activities such as nitric-oxide synthases
(26, 27) and antioxidant enzymes (28). GATA-4 may play a role in
promoting cell survival, as the apoptosis of ovarian cells was found to
be associated with a decrease in the expression of GATA-4 (29).
Furthermore, a lack of GATA-4 is also associated with activation of
apoptosis in the presumptive foregut (30). We recently found that the apoptosis of cardiac myocytes induced by anthracycline is associated with decreased GATA-4 expression and that the forced expression of
GATA-4 or -6 attenuates apoptosis, indicating that GATA-4 is involved
in cell survival signaling in cardiac myocytes (31).
To gain insights to the mechanism of HGF-mediated survival signaling in
cardiac myocytes, the present study explored the effects of HGF on
GATA-4. Results show that HGF activates GATA-4 in HL-1 cells and in the
primary culture of adult rat cardiac myocytes via
MEK/ERK-dependent phosphorylation, and this signaling
pathway is involved in HGF-mediated antiapoptotic responses.
Culture of Cardiac Muscle Cells--
HL-1 cardiac muscle cells
(32) were obtained from Dr. William Claycomb (Louisiana State
University, New Orleans, LA). Cells were maintained in EXCELL 320 medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal
bovine serum (Invitrogen), 10 µg/ml insulin (Invitrogen), 20 µg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake
Placid, NY), 1 µM retinoic acid (Sigma), 100 µM norepinephrine (Sigma), 1% nonessential amino acid
supplements (Invitrogen), 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml amphotericin B (Sigma) in plastic
dishes, coated with 12.5 µg/ml fibronectin and 0.02% gelatin, in a
5% CO2 atmosphere at 37 °C. Cells were replenished with
fresh media every 2-3 days. For treatment, cells were starved in
EXCELL 320 medium supplemented only with nonessential amino acids,
penicillin, streptomycin, and amphotericin B for 18 h and treated
with human recombinant HGF (Sigma).
Cardiac myocytes were isolated from ventricles of adult male Lewis rats
(3-6 months old) using an enzymatic isolation technique previously
described (33). Viable myocytes were purified via a series of gravity
sedimentations in 0.2, 0.5, and 1 mM Ca2+, and
then placed in Dulbecco's modified Eagle's medium/F-12 medium. Ca2+-tolerant myocytes purified through these procedures
were >90% viable.
Adenovirus-mediated Gene Transfer--
Adenovirus-directed gene
transfer was implemented by adding 30 plaque forming units of
recombinant adenovirus. The culture medium was aspirated from the cell
culture growing in a 35-mm dish, and 0.5 ml of the fetal bovine
serum-free medium containing the recombinant adenovirus was added. 1.5 ml of growth medium was added following 2 h of culture and
maintained for 24-48 h before performing experiments. Adenovirus
constructs expressing GATA-4 (wild-type and mutants) and dominant
negative MEK1 were kindly provided by Dr. J. Molkentin (University of Cincinnati).
Nuclear Extraction--
To prepare nuclear extracts, cells were
washed in phosphate-buffered saline and incubated in 10 mM
Hepes (pH 7.8), 10 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM sodium fluoride, 0.1 mM sodium
orthovanadate, and 1 mM tetrasodium pyrophosphate for 15 min at 4 °C. Nonidet P-40 was then added at a final concentration of
0.6% (v/v). Samples were mixed vigorously, and centrifuged. Pelleted
nuclei were resuspended in 50 mM Hepes (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA,
0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin,
10 µg/ml aprotinin, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium
pyrophosphate, and 1% (v/v) glycerol, then mixed for 20 min and
centrifuged for 5 min. The supernatants were harvested, protein
concentrations determined, and supernatants were stored at
Electrophoretic Mobility Shift Assay (EMSA)--
To perform
EMSA, binding reaction mixtures containing 2 µg of protein of nuclear
extract, 1 µg of poly(dI-dC)·poly(dI-dC), and
32P-labeled double stranded oligonucleotide probe
containing consensus GATA sequence
(5'-CACTTGATAACAGAAAGTGATAACTCT-3') in 100 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10% (v/v) glycerol, and 20 mM Tris-HCl (pH
7.5) were incubated for 20 min at 25 °C. Electrophoresis of samples
through a native 6% polyacrylamide gel was followed by autoradiography.
Western Blot Analysis--
SDS-PAGE gels (10%) used to separate
phosphorylated from nonphosphorylated GATA-4 contained acrylamide/bis
at a ratio of 30:0.165. Nuclear extracts (20 µg of protein) were
electrophoresed and electroblotted onto polyvinylidene difluoride
membranes. The membranes were blocked and incubated with rabbit
polyclonal IgG for GATA-4 (H-112) (Santa Cruz Biotechnology) at a
concentration of 1 µg/ml. Levels of proteins were detected with
horseradish peroxidase-linked secondary antibodies and ECL (enhanced
chemiluminescence) system (Amersham Biosciences). To determine the
effects of Comet Assay--
The neutral comet assay was employed to measure
double stranded DNA breaks as indication of cardiac myocyte apoptosis
(16). Treated cells were embedded in situ in 1% agarose,
then placed in lysis solution (2.5 M NaCl, 1% Na-lauryl
sarcosinate, 100 mM EDTA, 10 mM Tris base, 1%
Triton X-100) for 30 min. The nuclei were subsequently electrophoresed
for 20 min at 1 V/cm, followed by staining with SYBR Green and
visualized with a fluorescence microscope. Between 100 and 150 comets
per treatment were scored and assigned into type A, B, or C categories,
based on their tail lengths (34). Type C comets were defined as
apoptotic cells.
Statistical Analysis--
Mean ± S.E. were calculated, and
statistically significant differences between two groups were
determined by the Student's t test at p < 0.05.
HGF Induces GATA-4 Activation--
HGF has been shown to protect
cardiac myocytes against apoptotic death (15, 16). Because our recent
study (31) has shown that GATA-4 serves as a cell survival factor of
cardiac myocytes, we examined the effects of HGF on GATA-4. We found
that HGF increases the GATA DNA-binding activity. As shown in Fig.
1A, nuclear extracts from
cultured HL-1 cardiac muscle cells had some constitutive binding
activity toward a double stranded oligonucleotide containing the
consensus GATA sequence as determined by EMSA. This binding activity
was further increased by the treatment of cells with HGF (25 ng/ml).
The stimulation of GATA DNA-binding activity occurred within 3 min of
HGF treatment, and the densitometry analysis determined that the
intensity of the band increased 4-fold after 10 min of HGF treatment
(Fig. 1A, bar graph). Both constitutive and
activated GATA bands were effectively eliminated by cold double
stranded oligonucleotide competitors containing the GATA consensus
sequence in a concentration-dependent fashion (Fig.
1B). As shown in the line graph in Fig.
1B, the densitometry analysis revealed that, at a given
amount of cold competitor, the GATA-binding activity in HGF-treated
cells (closed squares) was higher than that in untreated
cells (open circle).
To determine the exact transcription factor species in HL-1 cells that
binds to the oligonucleotide containing the GATA consensus sequence and
is activated in response to HGF treatment, we performed supershift
experiments using GATA-4 and GATA-6 antibodies. As shown in Fig.
1C, both the constitutive and activated GATA complexes were
completely supershifted with the GATA-4 antibody whereas the GATA-6
antibody had no effects.
Similarly, HGF-activated GATA DNA-binding activity in primary cultures
of adult rat cardiac myocytes (Fig. 1D) in which GATA-4 is
the major GATA-binding protein (31). In the same nuclear extract
samples, we observed that the DNA-binding activity of NFAT (a known
mediator of GATA-4 activation) was not affected by HGF (Fig.
1D, lower panel). These results indicate that HGF activates the DNA-binding activity of GATA-4 in adult cardiac myocytes.
In contrast, other known cell survival factors such as NF- HGF Induces GATA-4 Phosphorylation--
We also noticed that the
complex between DNA and the HGF-activated GATA-4 migrated slightly
slower through the native gel compared with the complex with the
unstimulated GATA-4. Western blot analysis detected two GATA-4 bands at
~50 kDa in untreated HL-1 cells (Fig.
2A). The lower band was
slightly denser than the higher band (see densitometry analysis shown
in the bar graph of Fig. 2A). Treatment of cells
with HGF resulted in a time-dependent upward shift of the
lower band. By 10 min of treatment with HGF, all of the GATA-4 protein
molecules existed as a single species with a reduced mobility, and this
was sustained for at least 30 min.
To test the hypothesis that GATA-4 may be phosphorylated and thus give
a species with reduced mobility, we incubated nuclear extracts from
untreated and HGF-treated HL-1 cells with Role of MEK/ERK in HGF-induced GATA-4
Phosphorylation--
Towatari et al. (35) reported that
interleukin-3 activated GATA-2 through the ERK pathway in hematopoietic
progenitor cells. The GATA-4 molecule contains putative ERK
phosphorylation sites (36), and recent studies showed that hypertrophic
stimuli such as
To confirm the effects of these MEK inhibitors, we used adenovirus
expressing the dominant negative MEK described by Liang et
al. (40). As shown in Fig. 3C, HGF-induced activation
of ERK was blocked by dominant negative MEK expression without altering the ERK protein levels. Expression of this dominant negative mutant abolished the phosphorylation and activation of GATA-4 induced by HGF
(Fig. 3D).
Liang et al. (40) reported that GATA-4 is phosphorylated at
serine residue 105, an ERK phosphorylation site, in response to
phenylephrine treatment of neonatal ventricular myocytes. Consistently, the mutant GATA-4 with serine 105 replaced with alanine was not phosphorylated or activated by HGF in HL-1 cells (Fig.
4A) nor in primary culture of
adult rat cardiac myocytes (Fig. 4B), whereas endogenous
(Fig. 4A) or exogenous (Fig. 4B) wild-type GATA-4
was activated by HGF. Taken together, these results demonstrate that HGF induces phosphorylation and activation of GATA-4 via MEK/ERK phosphorylation of serine 105.
Role of MEK/ERK-dependent Phosphorylation of
GATA-4 in HGF-mediated Cell Survival--
HGF has been reported to
stimulate antiapoptotic Bcl-xL protein expression in a
human epithelial cell line (41) and cardiomyocytes (15). Similarly, HGF
up-regulated the expression of Bcl-xL in HL-1 cells (Fig.
5). To examine the role of the MEK/ERK
pathway in the up-regulation of Bcl-xL, the adenovirus
expressing a dominant negative mutant of MEK was employed. As shown in
Fig. 5, the dominant negative mutant of MEK blocked the HGF-induced
Bcl-xL expression.
To determine the role of GATA-4 in HGF-induced Bcl-xL
expression, a dominant mutant of GATA-4 as described by Liang et
al. (42) was used. EMSA experiments demonstrate that the
expression of this dominant negative mutant GATA-4 indeed inhibited
that GATA-4 DNA-binding activity in HL-1 cells (Fig.
6A). Reverse transcriptase-PCR analysis was used to monitor levels of bcl-x mRNA. The
data were quantified by calculating the ratio of bcl-x to
glyceraldehyde-3-phosphate dehydrogenase mRNA levels. As shown in
Fig. 6B, a dominant negative mutant GATA-4 increased the
basal level of bcl-x, but inhibited the HGF-induced
enhancement of bcl-x mRNA expression. Furthermore, forced expression of wild-type GATA-4 induced the expression of Bcl-xL (Fig. 6C), supporting the experiments
with dominant negative mutants demonstrating that GATA-4 regulates the
bcl-x expression.
To determine the role of serine 105 phosphorylation of GATA-4 in cell
survival, adenovirus expressing the S105A mutant of GATA-4 was used.
Unlike wild-type GATA-4, the S105A mutant did not cause enhancement of
Bcl-xL expression (Fig.
7A). Interestingly, we found
that this mutant serves as a dominant negative mutant. As demonstrated
in Fig. 7B, HGF inhibited the apoptotic cell death caused by
an anthracycline antibiotic, daunorubicin, but S105A mutant expression
blocked the HGF-induced protection of cells against daunorubicin. These
results suggest that HGF-mediated protection of cardiac muscle cells
is, at least partly, elicited by the up-regulation of antiapoptotic
Bcl-xL mediated by phosphorylation of GATA-4 at serine
105.
HGF has been shown to be released in response to myocardial
ischemia-reperfusion injury (10) and acute myocardial infarction (11,
12). Human serum HGF has been reported to increase from 0.3 to 0.4 ng/ml to a level as high as 37 ng/ml after acute myocardial infarction
(11). Studies using rat models have supported a protective role by
demonstrating that gene transfection (14) or injection (15) of HGF
attenuated ischemia-reperfusion injury in the heart. Therefore, HGF
appears to play an important role as an endogenous cardioprotective
factor. Furthermore, HGF may be useful as a therapeutic agent to
protect the heart against various oxidative stress stimuli. Thus, it is
important to understand the molecular mechanism of HGF action in the heart.
Some of the cardioprotective effects of HGF may be attributed to its
ability to induce angiogenesis by acting on vascular endothelial cells
(43). However, HGF may also directly affect cardiac myocytes by
attenuating the oxidative stress damage. In support of this hypothesis,
we have recently demonstrated that HGF directly protected adult cardiac
myocytes against oxidative stress stimuli such as serum deprivation,
H2O2, and daunorubicin (16). These results
suggest that the mechanism of HGF action to protect the heart may, in
part, involve its direct action on cardiac muscle cells to prevent the
occurrence of cell death.
HGF has also been shown to inhibit the apoptosis in noncardiac tissues.
Revoltella et al. (44) reported that C2.8 mouse embryonic
hepatocytic cells require exogenous HGF to survive and proliferate in
serum-free medium. Apoptosis of hepatocytes induced by interferon- Recently, Nakamura et al. (15) reported that HGF activated
the signal transduction pathway for increased Bcl-xL
expression, but not the phosphatidylinositol
3-kinase-dependent pathway, in intact hearts and in
cultured cardiomyocytes. The Bcl-xL expression was also
enhanced in HL-1 cells in response to HGF treatment. Previous studies
reported that Bcl-xL expression involves NF- In addition to the established role of GATA-4 in cardiac hypertrophy,
GATA-4 may also play a role in cell survival. We have recently shown
that the mechanism of anthracycline-induced cardiac muscle cell
apoptosis involves the down-regulation of GATA-4 (31). Gregory et
al. (24) demonstrated that GATA-1 induced the expression of
Bcl-xL, and the bcl-x gene has two GATA
consensus motifs that reside in the 5' promoter region (23). The
present study shows that GATA-4 is involved in HGF-mediated
antiapoptotic signaling.
Mechanisms of GATA-4 regulation may involve post-translational
modifications. A member of the GATA family, GATA-2, has been shown to
be phosphorylated in noncardiac muscle cells through the ERK pathway
(35). GATA-4 also contains putative ERK phosphorylation sites (36). It
has been demonstrated that hypertrophic stimuli, such as phenylephrine
(37) and endothelin-1 (38) induced a phosphorylation of GATA-4 in
cardiac myocytes. Recently, Liang et al. (40) demonstrated
that GATA-4 is phosphorylated at serine 105 by ERK2 in cardiomyocytes
in response to agonist stimulation, suggesting that GATA-4 might
regulate its downstream events via phosphorylation mediated by ERK1/2.
Indeed, our observations suggest that GATA-4 phosphorylation induced by
HGF leads to the up-regulation of Bcl-xL resulting in
HGF-mediated cardioprotection.
In summary, the present study examined the effects of HGF on GATA-4 in
cardiac muscle cells. As illustrated in Fig.
8, HGF activates GATA-4 whereas the
activation of other cell survival factors such as NF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain and a 34-kDa
-chain (1). The biologic activity of HGF as a potent mitogen of hepatocytes was first
demonstrated in the sera of normal and partially hepatectomized rats
(2, 3). HGF has been purified, cloned, and sequenced (1, 4). The HGF
receptor was identified as the c-met proto-oncogene that is
translated to a protein product, c-Met (5, 6). Signal transduction
pathways for HGF in these cells involve tyrosine phosphorylation of
c-Met and subsequent activation of p21ras (7), ERK (8), and
phosphatidylinositol 3-kinase (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
-protein phosphatase (
PPase), nuclear extracts were
isolated without the use of phosphatase inhibitors (sodium fluoride,
sodium orthovanadate, and tetrasodium pyrophosphate). Nuclear extracts
were incubated with 2,500 units/ml
PPase (New England Biolabs) at
30 °C for 30 min before subjecting to SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HGF enhances GATA-4 DNA-binding
activity. A, HL-1 cells were treated with HGF (25 ng/ml) for the durations as indicated. Nuclear extracts were prepared
and subjected to EMSA with the 32P-labeled double stranded
oligonucleotide containing the consensus GATA binding sequence. The
bar graph indicates the intensities of GATA bands in an
arbitrary unit as determined by densitometry. Values represent
mean ± S.E. (n = 4). An asterisk (*)
denotes the value that is significantly different from the untreated
control value at p < 0.05. B, nuclear
extracts from cells untreated or treated with HGF for 5 min were
subjected to EMSA with 32P-labeled oligonucleotide
containing the GATA binding sequence in the presence of increasing
amounts of a cold competitor with the GATA consensus sequence in the
binding reaction mixtures. The line graph indicates the
intensity of GATA bands in untreated (open circles) and
HGF-treated (closed squares) cells in an arbitrary unit as
determined by densitometry. C, HL-1 cells were treated with
HGF (25 ng/ml) for 5 min. Nuclear extracts from untreated and
HGF-treated cells were prepared and subjected to EMSA with
32P-labeled oligonucleotide containing the consensus GATA
sequence in the absence and presence of antibodies for GATA-4 or GATA-6
(2 µg) in the binding reaction mixtures. The arrow
indicates supershifted bands. D, primary culture of adult
rat cardiac myocytes were treated with HGF (25 ng/ml) for 10 min.
Homogenates were subjected to EMSA with 32P-labeled
oligonucleotide containing GATA or NFAT sequence. E, HL-1
cells were treated with a known activator of NF- B, tumor necrosis
factor-
(1 nM) for 1 h or HGF (25 ng/ml) for the
durations as indicated. Nuclear extracts were prepared and subjected to
EMSA with 32P-labeled oligonucleotide containing the
consensus NF-
B sequence.
B (Fig.
1E) and Akt (data not shown) are not activated by HGF.
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Fig. 2.
HGF induces the phosphorylation of
GATA-4. A, HL-1 cells were treated with HGF (25 ng/ml)
for the durations as indicated. Nuclear extracts were subjected to
reducing SDS-PAGE and Western blot analysis using the antibody against
GATA-4. Two GATA-4 bands identified at ~50 kDa in untreated cell
samples are indicated by arrows. The bar graph
indicates the ratio of lower to upper bands as determined by
densitometry analysis. B and C, nuclear extracts
from cells treated with HGF (25 ng/ml) were incubated in
vitro at 30 °C without or with PPase (2,500 units/ml).
Samples were subjected to SDS-PAGE and Western blot analysis using the
antibody for GATA-4 (B) and EMSA using the oligonucleotide
containing GATA binding sequence (C).
PPase in vitro.
For these experiments, we isolated nuclear extracts without the use of
phosphatase inhibitors. As shown in the Western blot results in Fig.
2B, the lower GATA-4 band shifted to a higher band in
response to treatment with HGF (first and second
lanes). This upward shift was slightly attenuated by incubating
the nuclear extracts at 30 °C (third and
fourth lanes), presumably because of actions of
endogenous protein phosphatases. The treatment of nuclear extract
samples from untreated and HGF-treated HL-1 cells with
PPase at
30 °C completely abolished the higher band (fifth and
sixth lanes). Similarly,
PPase treatment completely
abolished the upward shift of the GATA-4 band induced by HGF in EMSA
experiments (Fig. 2C). These results suggest that HGF
induced the phosphorylation of GATA-4 in HL-1 cells.
1-adrenergic agonist (37) and
endothelin-1 (38) induced the phosphorylation of GATA-4 in cardiac
myocytes. We, therefore, examined whether HGF phosphorylated GATA-4
through the MEK-ERK pathway. EMSA experiments showed that pretreatment
of cells with a specific inhibitor of MEK, PD98059 (39), effectively
blocked the upward shift (phosphorylation) of the GATA-4 band induced by HGF (Fig. 3A). Furthermore,
these results revealed that HGF-mediated enhancement of the GATA-4
DNA-binding activity was also blocked by the MEK inhibitor (Fig.
3A, bar graph). PD98059 was dissolved in Me2SO
and an equal amount of Me2SO (0.2% final concentration) alone had no effect (data not shown). Similarly, U0126 (another inhibitor of MEK) blocked the phosphorylation and activation of GATA-4
induced by HGF (data not shown). Western blot experiments confirmed our
observations that PD98059 abolished the upward mobility shift induced
by HGF (Fig. 3B). Unlike in vitro treatment with
PPase (Fig. 2B), a cell treatment with PD98059 was not
effective in eliminating the constitutively existing higher band and
only diminished the HGF-inducible phosphorylation.
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Fig. 3.
Role of MEK in HGF-induced GATA-4
activation. A, HL-1 cells were pretreated with 30 µM PD98059 (PD) for 30 min, then treated with HGF (25 ng/ml). Nuclear extracts were prepared and subjected to EMSA with
oligonucleotide containing the GATA binding sequence. The bar
graph indicates the intensity of GATA bands as determined by
densitometry. Values represent mean ± S.E. (n = 4). An asterisk (*) denotes the value that is significantly
different from the untreated control value at p < 0.05. B, HL-1 cells were pretreated with 30 µM
PD98059 for 30 min, then treated with HGF (25 ng/ml) for 10 min.
Nuclear extracts were subjected to Western blot analysis using the
antibody for GATA-4. C, HL-1 cells were infected with
adenovirus expressing dominant negative MEK (AdDN-MEK; 30 plaque-forming units/cell) for 48 h, then treated with HGF for 5 min. Cell lysates were subjected to Western blot analysis using the
phospho-specific ERK antibody. The membrane was stripped and re-blotted
with the ERK protein antibody (lower panel). D,
HL-1 cells were infected with adenovirus expressing dominant negative
MEK for 48 h, then treated with HGF for 10 min. Nuclear extracts
were prepared and subjected to EMSA with oligonucleotide containing
GATA binding sequence.
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Fig. 4.
Role of ERK-dependent
phosphorylation in HGF-induced GATA-4 activation.
A, HL-1 cells were infected with adenovirus expressing
GATA-4 mutant with serine 105 replaced with alanine (S105A) for 48 h, then treated with HGF (25 ng/ml) for 10 min. GATA-4 DNA-binding
activity was monitored by EMSA. B, primary culture of adult
rat cardiac myocytes were infected with adenovirus expressing wild-type
(WT) GATA-4 or GATA-4 mutant with serine 105 replaced with
alanine (S105A) for 48 h, then treated with HGF for 10 min. GATA-4
DNA-binding activity was monitored by EMSA.
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Fig. 5.
Role of MEK in HGF-induced expression of
Bcl-xL. Cells were infected with adenovirus expressing
dominant negative MEK (AdDN-MEK; 30 plaque-forming units/cell) for
48 h, then treated with HGF (25 ng/ml) for 24 h. Cell lysates
were prepared and subjected to Western blot analysis using the
Bcl-xL antibody. The bar graph indicates the
intensities of Bcl-xL bands as determined by densitometry.
Values represent mean ± S.E. (n = 4). An
asterisk (*) denotes the value that is significantly
different from the untreated control value at p < 0.05.
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Fig. 6.
Role of GATA-4 in HGF-induced expression of
Bcl-xL. A, HL-1 cells were infected with
control adenovirus (AdCont) without functional insert or
adenovirus expressing a dominant negative GATA-4 mutant
(AdDN-GATA4) for 48 h. Nuclear extracts were prepared
and the GATA DNA-binding activity was monitored by EMSA. B,
HL-1 cells were infected with control adenovirus or adenovirus
expressing a dominant negative GATA-4 mutant for 48 h, then
treated with HGF (25 ng/ml) for the durations as indicated. Total RNA
was isolated and subjected to reverse transcriptase-PCR analysis for
bcl-x and glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) mRNAs. The line graph shows -fold
increase in the bcl-x:glyceraldehyde-3-phosphate
dehydrogenase ratio in response to HGF treatment as determined by
densitometry (open circle, AdCont; closed square,
AdDN-GATA4). C, HL-1 cells were infected with control
adenovirus or adenovirus expressing wild-type GATA-4 for 48 h.
Cell lysates were prepared and subjected to Western blot analysis using
the Bcl-xL antibody. The membrane was re-blotted with the
antibody for GATA-4 (lower panel).
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Fig. 7.
Role of serine 105 of GATA-4 in
HGF-induced expression of Bcl-xL. A, HL-1
cells were infected with control adenovirus (AdCont) without
functional insert or adenovirus expressing wild-type GATA-4
(AdGATA4 wt) or GATA mutant (S105A) for 48 h. Cell lysates were prepared and subjected to Western blot analysis
using the Bcl-xL antibody. The bar graph
indicates the intensity of Bcl-xL bands as determined by
densitometry. Values represent mean ± S.E. An asterisk
(*) denotes the value that is significantly different from the value in
AdCont expressing cells at p < 0.05. B,
HL-1 cells were infected with control adenovirus or adenovirus
expressing a GATA-4 mutant (S105A) for 24 h, pretreated with HGF
(25 ng/ml) for 2 h, then treated with daunorubicin
(DNR; 1 µM) for 24 h. Apoptotic cells
were identified using the neutral comet assay. Values represent
mean ± S.E. (n = 4). An asterisk (*)
denotes the value that is significantly different from the value in
DNR-treated, control adenovirus-infected cells at p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was inhibited by HGF (45). In human endometrial epithelial cells, HGF
suppressed apoptosis induced by Fas antigen (46). A protective role for
HGF in acute renal failure is suggested because HGF protected renal
epithelial cells against apoptosis induced by serum starvation (47) or
cisplatin (48). Liu (41) reported that HGF triggered a phosphorylation
and resultant inactivation of pro-apoptotic Bad via the
phosphatidylinositol 3-kinase/Akt pathway and simultaneously
up-regulated antiapoptotic Bcl-xL. HGF was shown to block
the induction of apoptosis by various DNA damaging agents in breast
cancer cells via preventing down-regulation of Bcl-xL (49).
Thus, at least two pathways are responsible for antiapoptotic action of
HGF: (i) phosphatidylinositol 3-kinase-dependent inactivation of Bad and (ii) up-regulation of Bcl-xL
through unknown mechanisms.
B activation
(50). However, in our studies, HGF failed to activate this
transcription factor. Thus, NF-
B does not appear to play an
important cardioprotective role in the HGF-mediated activation of
Bcl-xL expression.
B and Akt was
not observed. HGF-induced activation of GATA-4 DNA-binding activity is
regulated by the MEK-ERK pathway-dependent phosphorylation.
Furthermore, our results demonstrate that the phosphorylation of GATA-4
plays an important role in the expression of antiapoptotic
Bcl-xL. We propose that the MEK/ERK/GATA-4 pathway is
involved in the HGF-mediated protection of cardiac myocytes against
oxidative stress-induced apoptosis, which can occur during myocardial
infarction.
View larger version (32K):
[in a new window]
Fig. 8.
A proposed model for the mechanism of
HGF-induced cardiac myocyte survival. The signal from HGF (via
c-Met tyrosine kinase receptor) activates MEK-ERK pathway which, in
turn, phosphorylates GATA-4. The phosphorylated GATA-4 up-regulates the
expression of anti-apoptotic proteins such as Bcl-xL
and prevents oxidative stress-induced cardiac myocyte apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Jane Remeika, Sarah Fitch, and Chia Chi Tan for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AG16121 (to Y. J. S.) and HL64282 (to T. E.) and the American Heart Association New England Affiliate (to Y. J. S.). This material is based upon work supported by U.S. Department of Agriculture under cooperative agreement 58-1950-9-001.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.
§ Visiting scientist from the National Food Research Institute of Japan, and received a thesis Ph.D. degree from Tohoku University based on the present work.
Recipient of the Career Development Award from the American
Heart Association National Center.
To whom correspondence should be addressed: USDA Human
Nutrition Research Center on Aging, Tufts University, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3148; Fax: 617-556-3344; E-mail:
yuichiro.suzuki@tufts.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M211616200
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ABBREVIATIONS |
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The abbreviations used are:
HGF, hepatocyte growth factor;
EMSA, electrophoretic mobility shift assays;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
PPase,
-protein phosphatase.
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