From the Departments of Pharmacology and Toxicology
and ¶ Pharmaceutical Sciences, University of Arkansas for Medical
Sciences, Little Rock, Arkansas 72205
Received for publication, December 4, 2002, and in revised form, February 10, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin (IL)-6 decreases
cardiac contractility via a nitric oxide (NO)-dependent
pathway. However, mechanisms underlying IL-6-induced NO production
remain unclear. JAK2/STAT3 and ERK1/2 are two well known
signaling pathways activated by IL-6 in non-cardiac cells. However,
these IL-6-activated pathways have not been identified in adult cardiac
myocytes. In this study, we identified activation of these two pathways
during IL-6 stimulation and examined their roles in IL-6-induced NO
production and decrease in contractility of adult ventricular myocytes.
IL-6 increased phosphorylation of STAT3 (at Tyr705)
and ERK1/2 (at Tyr204) within 5 min that peaked at 15-30
min and returned to basal levels at 2 h. Phosphorylation of STAT3
was blocked by genistein, a protein tyrosine kinase inhibitor, and
AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor.
The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but
not AG490. Furthermore, IL-6 enhanced de novo synthesis of
iNOS protein, increased NO production, and decreased cardiac
contractility after 2 h of incubation. These effects were blocked
by genistein and AG490 but not PD98059. We conclude that IL-6
activated independently the JAK2/STAT3 and ERK1/2 pathways, but only
JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.
IL-6,1 a
pro-inflammatory cytokine, is produced during the acute phase of the
immune response by macrophages, T cells, B cells, and non-immune cells
such as endothelial cells (1). After binding to its receptor, IL-6
elicits numerous effects including antibody induction, hematopoiesis,
thrombocytopoiesis, and acute-phase protein synthesis (1, 2).
Significant increases in serum levels of IL-6 and its mRNA and
protein expression in cardiac tissues have been reported in patients
with several cardiac diseases including congestive heart failure
(3-5), myocarditis (3), septic cardiomyopathy (6), myocardial
infarction (7-9), cardiac myxoma (10), and the cell injury associated
with ischemia/reperfusion (11) and cardiopulmonary bypass (12). Thus,
IL-6 has been suggested to play an important role in the
pathophysiology of these cardiac disorders.
In vitro studies in papillary muscle isolated from hamster
heart showed that IL-6 decreases contractility via a nitric oxide (NO)-dependent pathway during a 20-min exposure (13). IL-6
was also shown to decrease peak cytosolic intracellular
Ca2+ ([Ca2+]i) and
cell contraction of chick embryonic cardiomyocytes within minutes (14).
The acute IL-6-induced suppression of cardiac contractility and
[Ca2+]i was suggested to result
from activation of Ca2+-dependent NOS,
presumably a constitutive endothelial isoform (eNOS) (14). In the same
study, IL-6 was shown to induce iNOS expression after a 24-h
incubation, and it was suggested that the enhanced iNOS is responsible
for the chronic effect of IL-6 on the Ca2+ transient and
cell contraction (14). However, the signaling mechanism by which IL-6
activates iNOS and decreases cardiac contractility remains undefined.
IL-6 has been shown to activate both the JAK2/STAT3 pathway (15, 16)
and the ERK1/2 pathway through gp130, a signal transducing receptor, in
non-cardiac cells (15, 17, 18). For example, studies in liver and
neuronal cells have shown that IL-6 induces Tyr705
phosphorylation of STAT3, a transcription factor, via activation of
JAK2 (17, 19, 20). Activated STAT3 translocates to the nucleus and
activates expression of many genes in response to cytokines (21-24).
The role of gp130 in cardiac function has also been examined. One study
showed that transgenic mice with overexpression of gp130 displayed
hypertrophied ventricular myocardium (25). Studies in fetal murine
cardiac myocytes infected with adenovirus carrying wild-type or mutated
STAT3 cDNA suggested that the STAT3-dependent signaling
pathway plays a role in promoting the hypertrophy induced by leukemia
inhibitory factor (LIF), a member of IL-6-related cytokines (26). In
addition, activation of the JAK/STAT signaling pathway in rat heart has
been associated with the cardiac hypertrophy stimulated by
cardiotrophin-1, another member of IL-6-related cytokines (27), and
with cardiac dysfunction during ischemia and reperfusion (28),
myocardial ischemia (29), and acute myocardial infarction (30).
Although different isoforms of JAK/STAT have been associated with
effects of IL-6-related cytokines in adult rat heart, direct
links between IL-6 stimulation, activation of JAK/STAT, and
IL-6-induced cardiac inotropic actions have not been established.
Studies in neonatal rat cardiac myocytes have also shown that ERK1/2 is
activated by LIF via a gp130-dependent process (31) and
that the ERK1/2 activation is associated with cardiac hypertrophy (32).
In contrast, LIF-induced hypertrophy occurred only in fetal cardiac
myocytes infected with a wild-type STAT3 cDNA with the level of
ERK1/2 activation showing no difference from those cells infected with
mutated STAT3 cDNA (26). Thus, the functional role of
gp130-associated activation of ERK1/2 and JAK/STAT in IL-6-induced
cardiac effects remains unclear.
In this study, we first demonstrated that IL-6 activated both the
JAK2/STAT3 and ERK1/2 pathways in adult ventricular myocytes. We also
showed that protein expression of iNOS and an increase in NO production
were first detectable after 2 h of IL-6 stimulation. Such
treatment with IL-6 also decreased the contractile function of
ventricular myocytes. Most importantly, we found that the IL-6-induced activation of iNOS and decrease in contractility were mediated by
JAK2/STAT3, but not by ERK1/2.
Myocyte Isolation--
Single ventricular myocytes were isolated
from hearts of adult (3-6-month-old) male Sprague-Dawley rats using
enzymatic dissociation as described previously (33). Isolated cells
were plated into Petri or culture dishes (Falcon) containing a
serum-free, L-arginine-containing culture medium and
incubated overnight. Myocytes were then treated with 10 ng/ml IL-6 for
various periods of time.
The use of animals was carried out under a protocol approved by the
Animal Care and Use Committee at the University of Arkansas for Medical Sciences.
Western Blot Analysis--
Protein preparation and
immunoblotting were carried out as described previously (34). Briefly,
control and treated myocytes (0.25 × 106 cells/group)
were lysed in ice-cold lysis buffer containing 20 mM HEPES,
250 mM sucrose, 2 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, 10 mM sodium
orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 10 µg/ml aprotinin. The concentration of total
protein was determined using a Bradford assay (Bio-Rad). 20-40
µg of each protein sample were separated electrophoretically on 10%
SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. After blocking with Tris buffer solution containing 0.05%
Tween 20 and 5% non-fat milk for 1 h at room temperature, membranes were probed with polyclonal anti-phospho-STAT3 antibodies (1:1000 dilution; Cell Signaling Technology Inc., Beverly, MA), monoclonal anti-phospho-ERK antibodies (1:1,000 dilution; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), or polyclonal anti-iNOS antibodies (1:1,000 dilution; Cayman Co., Ann Arbor, MI) overnight at
4 °C. Immunoblots were detected using enhanced chemiluminescent kits
(SuperSignal; Pierce) and analyzed with a densitometer (Bio-Rad). The
membranes were then stripped and reprobed with polyclonal anti-STAT3
antibodies (1:2,000 dilution; Santa Cruz Biotechnology, Inc.) or
polyclonal anti-ERK antibodies (1:2,000 dilution; Santa Cruz
Biotechnology, Inc.).
Immunocytochemistry--
Myocytes were plated onto sterile
multi-chamber slides (Nalge Nunc Corp., Naperville, IL) in serum-free
culture media overnight at 37 °C. After treatment with 10 ng/ml
IL-6, cells were washed twice with PBS, fixed with 4% paraformaldehyde
for 10 min, and permeabilized with PBS containing 0.1% Triton X-100.
Cells were then incubated for 20 min in PBS containing 10% goat serum
(Jackson ImmunoResearch Laboratories, West Grove, PA), washed with PBS, and incubated for 60 min with polyclonal anti-iNOS antibodies (1:100;
Cayman Co., Ann Arbor, MI) in PBS containing 1.5% goat serum at room
temperature. After three washes with PBS (5 min each), cells were
incubated for 45 min in PBS containing fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:100; Jackson ImmunoResearch Laboratories) and 1.5% goat serum in a dark chamber and
then examined using an epifluorescent microscope (Zeiss).
NO Production--
NO production was assessed by
measuring total nitrate and nitrite (NOx) in culture
media and cell lysates. Myocytes were incubated in serum-free
and phenol-red-free culture medium in the absence and presence of 10 ng/ml IL-6 for 2, 4, and 24 h before being centrifuged at 100 × g for 10 min. The supernatant (culture media) was removed
and kept at Measurements of Cell Shortening (CS)--
After the designated
exposure duration, contraction of ventricular myocytes was elicited by
field stimulation (2-ms duration, 1.5-fold threshold voltage) at 0.5 Hz
in normal Tyrode's solution containing the following (in
mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2, 10 HEPES/Tris, and 5.6 glucose. CS was monitored
using an edge motion detector (Crescent Electronics, Sandy, UT) as
described previously (35). The voltage signal was calibrated to
determine actual motion (µm). Post-rest potentiation (PRP), which has
been used to assess cardiac Ca2+ handling and contractile
function (36, 37), was measured as the first contraction after given
rest intervals (e.g. for 30 s (PRP30) or 60 s
(PRP60)). The relative amplitude of PRP CS was presented by normalizing
its peak amplitude to that of steady-state CS before the rest interval.
Chemicals--
Recombinant rat IL-6 was purchased from Pepro
Tech Inc (Rocky Hill, NJ); the nitrate/nitrite colorimetric assay kit
was obtained from Cayman Co. Cycloheximide, genistein, PD98059, and
AG490 were purchased from Calbiochem.
Statistical Analysis--
In biochemical assays, all treated
groups were normalized to each time control and presented as means ± S.E. Statistical significance (p < 0.05) was
evaluated by Student's t test or analysis of variance with
Duncan's multiple range test.
IL-6-induced Phosphorylation of STAT3 in Adult Rat Ventricular
Myocytes--
Although activation of JAK/STAT signaling has been
associated with gp130-related stimulation by LIF (26) and
cardiotrophin-1 (27), there were no data demonstrating a direct link of
this pathway to IL-6 stimulation in cardiac myocytes. Thus, we first examined whether IL-6 activates the JAK2/STAT3 pathway in adult ventricular myocytes. Fig. 1A
shows that phosphorylation of STAT3 at Tyr705 was detected
after 5 min of exposure to 10 ng/ml IL-6, reached a maximum at 15 min,
and then declined within 1 h. Fig. 1B shows combined
data of the time course for the phosphorylation of STAT3, which was
increased 9.3-, 12.1-, 9.7-, and 7.6-fold (n = 4, p < 0.05, compared with time control) after 5, 15, 30, and 60 min of exposure to IL-6, respectively. The level of
phosphorylation returned to basal levels after 2 and 4 h of
treatment, and a second increase (~ 2-fold) was observed at 24 h
(Fig. 1B). Total STAT3 protein levels remained relatively
constant during the 24-h stimulation (lower panel in Fig.
1A). These results demonstrate that IL-6 activates a STAT3
signaling in adult rat ventricular myocytes.
Fig. 2 shows the effects of three
different kinase inhibitors on the phosphorylation of STAT3 after 30 min of exposure to 10 ng/ml IL-6. Cells were pretreated for 30 min with
these inhibitors before IL-6 exposure. IL-6-induced phosphorylation of
STAT3 at Tyr705 was blocked by 20 µM AG490, a
JAK2 inhibitor, and by 10 µM genistein, a protein
tyrosine kinase inhibitor, but not by 10 µM PD98059, an
ERK1/2 kinase inhibitor (Fig. 2, A and B). These
data suggest that IL-6-induced tyrosine phosphorylation of STAT3 is
mediated through JAK2 activation.
IL-6-induced Phosphorylation of ERK1/2 in Adult Rat
Ventricular Myocytes--
Fig.
3A shows that an increase in
phosphorylation of ERK2 at Tyr204 was detected after 5 min
of exposure to 10 ng/ml IL-6; this effect peaked at 30 min and was
followed by a decline to basal levels by 2 h. Note that ERK2 is
the predominate form in adult ventricular myocytes, and the basal level
of p-ERK2 (each time control, as well as zero-time control) also
remained relatively constant. Fig. 3B summarizes combined
data showing a time-dependent IL-6-induced phosphorylation
of ERK2 with increases of 5.2-, 5.0-, 9.5-, and 5.1-fold (compared with
time controls) after 5, 15, 30, and 60 min of exposure
(n = 3, p < 0.05), respectively. The
level of ERK2 phosphorylation at 2, 4, and 24 h was not
significantly different from time controls. These results suggest that
IL-6 transiently activates ERK1/2 signaling in adult rat ventricular
myocytes.
Fig. 4 shows that after 30 min of
exposure to IL-6 the marked increase in phosphorylation of ERK2 at
Tyr204 was blocked by 10 µM PD98059 and 10 µM genistein but not by 20 µM AG490. These
data suggest that IL-6-induced phosphorylation of ERK1/2 is independent
of the JAK2/STAT3 signaling pathway.
Expression of iNOS Protein Induced by IL-6 in Adult Rat Ventricular
Myocytes--
Although iNOS expression was detected after 24 h of
IL-6 stimulation in chick embryonic heart cells (14), the time course of induction of iNOS has not been examined in adult cardiac myocytes. Fig. 5A shows the
time-dependent increase in iNOS protein (~130 kDa)
expression in response to 10 ng/ml IL-6 in adult rat ventricular myocytes. iNOS protein was undetectable at 1 h but was increased after 2, 4, and 24 h of incubation with IL-6. Fig. 5B,
which represents combined data from five experiments, shows that iNOS
protein expression in IL-6-treated myocytes was increased 9-fold,
compared with time controls after 2 h of treatment. Similarly,
immunocytochemical data in Fig. 5C show that iNOS was
evident in a myocyte after 2 h of exposure to 10 ng/ml IL-6,
whereas it was barely detectable in 2-h time control myocytes or after
1 h of IL-6 stimulation. The iNOS expression induced by a 2-h
incubation with 10 ng/ml IL-1
We then determined whether de novo protein synthesis is
involved in IL-6-induced iNOS protein expression. Fig.
6A shows that total
NOx concentration in culture medium was increased after a
2-h exposure to 10 ng/ml IL-6 (note that the IL-6-induced
NOx production was blocked completely by 50 nmol/liter
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine, a potent and selective
inhibitor of iNOS) (39). This 2.5-fold increase in NOx
production was abolished in the presence of 10 µM
cycloheximide (CHX), an inhibitor of protein synthesis that was applied
30 min prior to and during the IL-6 treatment. The NOx
concentration in the 2-h time control was 2.65 ± 0.46 nmol/mg cell protein and was not affected by CHX. Similarly, Fig. 6B
shows that under the same conditions of IL-6 treatment, the
NOx concentration in cell lysates was increased ~3.5-fold
of the time control after 2 h of incubation. CHX completely
blocked this IL-6-induced increase in NOx production and
had no effect on the basal level (2.18 ± 0.31 nmol/mg cell
protein) in cell lysate. These results suggest that de novo
protein synthesis and the activity of iNOS are required for
IL-6-induced NO production during a 2-h exposure.
JAK2/STAT3, Not ERK1/2, Mediates IL-6-induced
iNOS Protein Expression and NO Production--
Fig.
7 shows that the iNOS protein expression
induced 2 h after exposure to 10 ng/ml IL-6 was blocked completely
by pretreatment with 20 µM AG490 and 10 µM
genistein. In contrast, pretreatment with 10 µM PD98059
had no effect on IL-6-induced iNOS expression. Similar results were
observed in five experiments. Fig. 8
shows that IL-6 increased the total NOx concentration in
cell lysates ~2.4-fold (n = 5, p < 0.05) after 2 h of exposure when compared with time controls. This
IL-6-induced increase in NO production was blocked by AG490 and
genistein but not by PD98059. Similar results were observed in cell
lysate after a 4-h incubation (data not shown). These results suggest
that IL-6-induced iNOS protein expression and NO production are
mediated by activation of the JAK2/STAT3 pathway, but not the ERK1/2
pathway.
Role of JAK2/STAT3 Pathway and ERK1/2 Pathway
in IL-6-induced Decrease in Cardiac Contractility--
Our preliminary
findings showed that IL-6 decreased contractility in adult ventricular
myocytes as demonstrated by reductions in PRP and the responsiveness to
extracellular Ca2+ concentrations (39). Thus, we
examined whether the JAK2/STAT3 and/or ERK1/2 pathways are involved in
the IL-6-induced negative inotropic effect. Fig.
9A shows representative data
of PRP30 and PRP60 in a time-control (upper panel) and an
IL-6 treated myocyte (lower panel). The amplitude of PRP30
and PRP60 relative to that of pre-rest steady state is shown in Fig. 9,
B and C, respectively. Exposure for 2 h to
10 ng/ml IL-6 reduced PRP30 and PRP60 by ~34% (n = 17, p < 0.05) and 32% (n = 16, p < 0.05), respectively. The IL-6-induced decreases in
PRP30 and PRP60 were blocked by 20 µM AG490 and 10 µM genistein but not by 10 µM PD98059.
Pretreatment with PD98059, AG490, or genistein alone had no effect on
PRP30 or PRP60. The same results were observed after 4 and 24 h of
exposure to IL-6 (data not shown). These data suggest that the
IL-6-induced decrease in cardiac contractility is mediated by
activation of the JAK2/STAT3, but not ERK1/2, pathway.
Recently, both IL-6-related cytokines and the JAK/STAT signaling
pathway have been suggested to play important roles in cardiac pathophysiology (40). Our preliminary studies suggested that IL-6
decreases contractility of adult rat ventricular myocytes via an iNOS
pathway during chronic exposure (39), consistent with findings reported
by others using chick cardiomyocytes (14). The mechanism underlying
IL-6-induced iNOS has not been defined. In this study, we demonstrated
that IL-6 activates both the JAK2/STAT3 and ERK1/2 pathways in adult
ventricular myocytes. We also showed that 1) IL-6 induces iNOS protein
expression via de novo synthesis within 2 h,
accompanied by an increase in NO production in culture medium and cell
lysates, and 2) IL-6-induced iNOS expression, increase in NO
production, and decrease in contractility are blocked by inhibition of
STAT3 phosphorylation with a JAK2 inhibitor and a protein tyrosine
kinase inhibitor but not by inhibition of ERK1/2 activation.
IL-6-related cytokines, such as IL-6 and LIF, bind to distinct
receptors and trigger the formation of homodimerization of a common
signal transducing receptor, gp130, or heterodimerization of gp130 with
the LIF receptor (15). Activation of gp130 involves rapid tyrosine
phosphorylation as induced by LIF (31) or by IL-6 (41), which is
believed to be required for association with members of the JAK family
such as JAK1, JAK2, and Tyr-2 and recruitment of STAT3 to gp130 (15).
Thereafter, tyrosine-specific phosphorylation of STAT3 occurs and
initiates its downstream cascade to alter gene expression. Activation
of gp130 has also been shown to activate the Ras-mitogen-activated
protein kinase (or ERK1/2) cascade (31, 32). In the present study, we
showed that IL-6 enhances tyrosine phosphorylation of both STAT3 and
ERK1/2 in a similar time course and that both activations are blocked
by genistein. In contrast, a JAK2 inhibitor (AG490) blocked only IL-6-induced activation of STAT3 but not the increased ERK1/2 phosphorylation, whereas PD98058 blocked IL-6-induced activation of
ERK1/2 but not STAT3. These results suggest that 1) IL-6 activates the
JAK2/STAT3 and ERK1/2 pathways independently via tyrosine phosphorylation of g130 (e.g. blocked by genistein) in adult
ventricular myocytes, and 2) the JAK2/STAT3 activity is not required
for IL-6-induced activation of ERK1/2 and vice versa.
The time course of iNOS induction by cytokines in cardiac myocytes is
not clearly defined. For example, IL-6-induced iNOS expression was
detected in chick embryonic heart cells after a 24-h incubation (14).
However, IL-6 levels may be increased for relatively short periods
after insults such as cardiopulmonary bypass. The present study
examined the time course of IL-6-induced iNOS expression in adult
ventricular myocytes and found that de novo synthesis of
iNOS protein was detected within 2 h of IL-6 stimulation. Such
increased iNOS is accompanied by an increase in NO production and a
decrease in contractile function. These IL-6-elicited cardiac effects
were sustained during 24 h of exposure to the cytokine.
The iNOS gene promoter region has three sites for STAT binding (42).
Studies using human DLD-1 cells, a colon epithelial-derived cell line,
showed that a cytokine mixture of INF- The ERK1/2 pathway has been shown to play an important role in the
cardiac hypertrophy induced by a variety of stimuli. However, the role
of IL-6-induced increases in ERK1/2 phosphorylation in cardiac function
is still unclear. For example, gp130-dependent activation
of ERK1/2 induced by LIF induces hypertrophy in neonatal rat cardiac
myocytes (32). In contrast, LIF-induced ERK1/2 activation was observed
in fetal cardiac myocytes infected with a wild-type STAT3 cDNA and
mutated STAT3 cDNA, whereas only cells infected with intact STAT3
displayed hypertrophy (26). The same study also showed that LIF-induced
increase in STAT3-dependent c-fos and atrial
natriuretic factor mRNA expression was attenuated by inhibition of
ERK1/2 by PD98059, suggesting a cross-talk between JAK/STAT and ERK1/2
signaling in fetal murine cardiac myocytes (26). In contrast, the
present study showed that the iNOS expression, NO production, or
negative inotropy induced by IL-6 was not affected by inhibition of
ERK1/2. Although the role of IL-6-induced activation of ERK1/2 in
cardiac function remains unclear, it is clear that ERK1/2 activation is
not involved in the JAK2/STAT3 signaling pathway nor the negative
inotropic actions of IL-6 in adult ventricular myocytes. In contrast to
our results, activation of ERK1/2 has been shown to be essential to the
induction of iNOS expression elicited by a cytokine mixture of INF- In summary, both the JAK2/STAT3 and ERK1/2 pathways are activated
transiently by IL-6 in adult rat ventricular myocytes. Only the
activation of JAK2/STAT3 mediates the IL-6-induced increase in iNOS
protein expression, increase in NO production, and decrease in cardiac
contractility. Thus, the IL-6-activated JAK2/STAT3 signaling pathway
could account for the cardiac dysfunction observed in many
cytokine-associated cardiac disorders such as acute myocardial infarction, ischemia/reperfusion, myocarditis, or cardiopulmonary bypass.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until the NOx assay. The pellets were resuspended in 500 µl of PBS (pH 7.4), half of which was used to
determine protein content. The other half was sonicated and centrifuged
at 10,000 × g for 20 min. The resulting supernatant was centrifuged at 100,000 × g for 15 min at 4 °C,
and the final supernatant was filtered through a 10-kDa molecular
mass cut-off filter (Fisher Scientific, Pittsburgh, PA) at
12,000 × g for 30 min. These cell lysates were kept at
20 °C until the NOx assay. The concentration of
NOx in culture media and cell lysates was determined using
a colorimetric assay kit with a detection limit of 2.5 µM
(Cayman Co., Ann Arbor, MI) according to the manufacturer's instructions. Nitrate/nitrite concentrations were averaged from duplicate or triplicate measurements and reported as nmol
NOx per mg cell protein. Results of treated groups were
then normalized to each time control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 1.
Time course of phosphorylation of STAT3 in
response to IL-6 stimulation. A, a representative experiment
showing that phosphorylation of STAT3 (p-STAT3) in time
controls (C) and myocytes treated with 10 ng/ml IL-6
(I) for indicated times was detected with anti-phospho-STAT3
(Tyr705) antibodies (top panel). After stripping
phosphor-specific antibodies, total protein levels of STAT3 in the same
samples were detected using anti-STAT3 antibodies (lower
panel). B, phosphorylation level of STAT3 in each group
was presented as a ratio of phosphorylated to total STAT3
(p-STAT3/STAT3), which was then normalized to
each untreated time control. Data represent mean ± S.E. for three
to four experiments. *, p < 0.05, compared with time
control.
View larger version (38K):
[in a new window]
Fig. 2.
Effects of kinase inhibitors on IL-6-induced
tyrosine phosphorylation of STAT3. Myocytes were treated with 10 µM PD98059, 20 µM AG490, or 10 µM genistein alone for 1 h or 30 min before and
during exposure for 30 min to 10 ng/ml IL-6. A, a
representative experiment showing that IL-6-induced tyrosine
phosphorylation of STAT3 (p-STAT3; top panel) was
blocked by AG490 and genistein but not by PD98059. B,
combined data from three experiments showing p-STA3/STAT3 relative to
time controls. These inhibitors alone had no significant effect on
STAT3 phosphorylation. *, p < 0.05, compared with
control; #, p < 0.05, compared with PD98059
alone.
View larger version (29K):
[in a new window]
Fig. 3.
Time course of tyrosine phosphorylation of
ERK1/2 in response to IL-6 stimulation. A, phosphorylation
of ERK1/2 at Tyr204 (p-ERK1/2;
top panel) in the same cell lysates as shown in Fig. 1,
which was then immunoblotted for total ERK1/2 (lower panel).
B, combined data from three experiments showed the time
course of phosphorylation level of ERK2
(p-ERK2/ERK2) relative to time controls. *,
p < 0.05, compared with control.
View larger version (32K):
[in a new window]
Fig. 4.
Effects of kinase inhibitors on IL-6-induced
tyrosine phosphorylation of ERK2. A, an inhibition of
IL-6-induced tyrosine phosphorylation of ERK2 by PD98059 and genistein
in the same protein samples used for the detection of STAT3
phosphorylation. B, combined data from three experiments
showing relative p-ERK2/ERK2 to time controls. *, p < 0.05, compared with control; #, p < 0.05, compared
with AG490 alone.
, a well known inducer of iNOS in
cardiac myocytes (38), was used as a positive control in these
experiments. Thus, these results suggest that IL-6 induces iNOS protein
expression within 2 h of exposure.
View larger version (36K):
[in a new window]
Fig. 5.
Time course of iNOS protein expression in
response to IL-6 stimulation. A, Western blot
analyses of iNOS protein expression (at 130 kDa) in adult ventricular
myocytes treated for indicated times without (C, time
control) or with 10 ng/ml IL-6 (I). B, relative
density of iNOS protein expression in IL-6-treated cells (for 2 h)
from five experiments. *, p < 0.05, compared with time
control. C, immunocytochemical data showing fluorescent
images of iNOS protein expression (top panel) obtained from
cells in the absence (time control, left panel)
or presence of 10 ng/ml IL-6 for indicated times. Myocytes treated with
IL-1 for 2 h were used as positive control (right
panel). Bright-field images of the same cells were shown in the
lower panel.
View larger version (16K):
[in a new window]
Fig. 6.
Effect of cycloheximide on the IL-6-induced
increase in NO production. Myocytes were treated with and without
10 ng/ml IL-6 for 2 h in the absence and presence of 10 µM CHX, a protein synthesis inhibitor. Some cells were
treated with CHX alone for 2 h. A, normalized
concentrations of NOx in culture media were expressed as
ratios of the NOx concentration in treated groups to the
control value. B, NOx concentrations in cell
lysates obtained from the same experiments as shown in A.
Data represent means ± S.E. from three experiments. *,
p < 0.05, compared with the control.
View larger version (29K):
[in a new window]
Fig. 7.
Effects of kinase inhibitors on IL-6-induced
iNOS protein expression. Expression of iNOS protein was examined
in myocytes after 2 h of exposure to 10 ng/ml IL-6 in the absence
and presence of 10 µM PD98059, 20 µM AG490,
or 10 µM genistein as shown in Fig. 2. Cell lysate from
lipopolysaccharide-treated macrophage 264.7 cells served as positive
control. Similar results were observed in four other
experiments.
View larger version (24K):
[in a new window]
Fig. 8.
Effects of kinase inhibitors on IL-6-induced
increase in NO production. Cell lysates were obtained from
myocytes pretreated with 10 µM PD98059, 20 µM AG490, or 10 µM genistein for 30 min
before exposure to 10 ng/ml IL-6 for 2 h. Normalized
NOx concentrations of treated groups were presented as
ratio to each control value. The NO production in myocytes treated with
kinase inhibitors alone for 2.5 h did not differ from that in time
controls. Data represent means ± S.E. from three to eight
experiments. *, p < 0.05, compared with control; #,
p < 0.05, compared with PD98058 alone.
View larger version (39K):
[in a new window]
Fig. 9.
Effects of kinase inhibitors on IL-6-induced
decrease in post-rest potentiation. A, representative
cell shortening traces of PRP after 30 (PRP30)- and 60-s (PRP60) rest
intervals in a time control and a myocyte pretreated with 10 ng/ml IL-6
for 2 h. B, relative PRP30 (top panel) and
PRP60 (lower panel) normalized to the pre-rest steady-state
amplitude of cell shortening from time controls and myocytes pretreated
with PD98059, AG490, or genistein for 30 min before exposure to 10 ng/ml IL-6 for 2 h. These kinase inhibitors alone had no effect on
RP30 or PRP60 after 2.5 h of incubation. Data represent means ± S.E. from four to seventeen cells. *, p < 0.05, compared with control; #, p < 0.05, compared with
PD98058 alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-1
, and tumor necrosis
factor-
induced iNOS expression via activation of the JAK2/STAT1
pathway, which was blocked by AG490 (43). INF-
-induced iNOS
expression has also been shown to be paralleled by STAT1
activation
in adult rat ventricular myocytes (44). Therefore, it is likely that
STAT3 activation also mediates IL-6-induced iNOS protein synthesis.
In the present study, we showed that genistein and AG490 block
IL-6-induced STAT3 phosphorylation, iNOS protein expression, NO
production, and negative inotropy. These results are consistent with
the hypothesis that the IL-6-induced JAK2/STAT3 signaling pathway
mediates its induction of iNOS, thereby increasing NO production and
decreasing cardiac contractility.
and IL-1
in adult rat ventricular myocytes (44). This discrepancy
may result from activation of different STATs by different cytokines.
For example, the combination of INF-
and IL-1
activates STAT1
,
whereas IL-6 activates primarily STAT3. The activation of STAT1
might be associated with both ERK1/2 and JAK2 activation, whereas only
JAK2 is involved in STAT3 activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the expert technical assistance of Meei-Yueh Liu and Kerrey A. Roberto.
![]() |
FOOTNOTES |
---|
* This study was supported in part by grants from the Office of Naval Research and the American Heart Association/Heartland Affiliate and by NHLBI, National Institutes of Health Grant R01HL62226.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.
§ Recipient of a predoctoral fellowship from the American Heart Association/Heartland Affiliate and a grant from the University of Arkansas for Medical Sciences Graduate Student Research Fund.
To whom correspondence should be addressed: Dept. of
Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham St., MS #522-3, Little Rock, AR 72205. Tel.: 501-686-8106; Fax: 501-686-6057; E-mail: sliu@uams.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M212321200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
NO, nitric oxide;
NOS, nitric-oxide synthase;
iNOS, inducible NOS;
JAK, Janus kinase;
STAT, signal transducer and activator of transcription;
ERK, extracellular signal-regulated kinase;
CS, cell shortening;
PRP, post-rest potentiation;
LIF, leukemia inhibitory factor;
INF-, interferon-
;
PBS, phosphate-buffered saline;
CHX, cycloheximide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Akira, S., Hirano, T., Taga, T., and Kishimoto, T. (1990) FASEB J. 4, 2860-2867[Abstract] |
2. | Kammuller, M. E. (1995) Toxicology 105, 91-107[CrossRef][Medline] [Order article via Infotrieve] |
3. | Satoh, M., Tamura, G., Segawa, I., Tashiro, A., Hiramori, K., and Satodate, R. (1996) Virchows Arch. Int. J. Pathol. 427, 503-509 |
4. | Steele, I. C., Nugent, A. M., Maguire, S., Hoper, M., Campbell, G., Halliday, M. I., and Nicholls, D. P. (1996) Eur. J. Clin. Invest. 26, 1018-1022[Medline] [Order article via Infotrieve] |
5. | Roig, E., Orus, J., Pare, C., Azqueta, M., Filella, X., Perez-Villa, F., Heras, M., and Sanz, G. (1998) Am. J. Cardiol. 82, 688-690[CrossRef][Medline] [Order article via Infotrieve] |
6. | Hack, C. E., De Groot, E. R., Felt-Bersma, R. J., Nuijens, J. H., Strack, Van Schijndel, R. J., Eerenberg-Belmer, A. J., Thijs, L. G., and Aarden, L. A. (1989) Blood 74, 1704-1710[Abstract] |
7. | Ikeda, U., Ohkawa, F., Seino, Y., Yamamoto, K., Hidaka, Y., Kasahara, T., Kawai, T., and Shimada, K. (1992) J. Mol. Cell. Cardiol. 24, 579-584[Medline] [Order article via Infotrieve] |
8. | Guillen, I., Blanes, M., Gomez-Lechon, M. J., and Castell, J. V. (1995) Am. J. Physiol. 269, R229-R235[Medline] [Order article via Infotrieve] |
9. |
Neumann, F.-J.,
Ott, I.,
Gawaz, M.,
Richardt, G.,
Holzapfel, H.,
Jochum, M.,
and Schömig, A.
(1995)
Circulation
92,
748-755 |
10. | Jourdan, M., Bataille, R., Seguin, J., Zhang, X. G., Chaptal, P. A., and Klein, B. (1990) Arthritis Rheum. 33, 398-402[Medline] [Order article via Infotrieve] |
11. |
Sawa, Y.,
Ichikawa, H.,
Kagisaki, K.,
Ohata, T.,
and Matsuda, H.
(1998)
J. Thorac. Cardiovasc. Surg.
116,
511-517 |
12. |
Wan, S.,
DeSmet, J. M.,
Barvais, L.,
Goldstein, M.,
Vincent, J. L.,
and LeClerc, J. L.
(1996)
J. Thorac. Cardiovasc. Surg.
112,
806-811 |
13. | Finkel, M. S., Oddis, C. V., Jacob, T. D., Watkins, S. C., Hatttler, B. G., and Simmons, R. L. (1992) Science 257, 387-389[Medline] [Order article via Infotrieve] |
14. | Kinugawa, K., Takahashi, T., Kohmoto, O., Yao, A., Aoyagi, T., Momomura, S., Hirata, Y., and Serizawa, T. (1994) Circ. Res. 75, 285-295[Abstract] |
15. |
Kishimoto, T.,
Akira, S.,
Narazaki, M.,
and Taga, T.
(1995)
Blood
86,
1243-1254 |
16. | Hibi, M., Nakajima, K., and Hirano, T. (1996) J. Mol. Med. 74, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
17. | Schumann, G., Huell, M., Machein, U., Hocke, G., and Fiebich, B. L. (1999) J. Neurochem. 73, 2009-2017[Medline] [Order article via Infotrieve] |
18. |
Ueda, T.,
Bruchovsky, N.,
and Sadar, M. D.
(2002)
J. Biol. Chem.
277,
7076-7085 |
19. | Debonera, F., Aldeguer, X., Shen, X. D., Gelman, A. E., Gao, F., Que, X. Y., Greenbaum, L. E., Furth, E. E., Taub, R., and Olthoff, K. M. (2001) J. Surg. Res. 96, 289-295[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Niehof, M.,
Streetz, K.,
Rakemann, T.,
Bischoff, S. C.,
Manns, M. P.,
Horn, F.,
and Trautwein, C.
(2001)
J. Biol. Chem.
276,
9016-9027 |
21. | Decker, T. (1999) Cell. Mol. Life Sci. 55, 1505-1508[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Kiuchi, N.,
Nakajima, K.,
Ichiba, M.,
Fukada, T.,
Narimatsu, M.,
Mizuno, K.,
Hibi, M.,
and Hirano, T.
(1999)
J. Exp. Med.
189,
63-73 |
23. | Schindler, C. (1999) Exp. Cell Res. 253, 7-14[CrossRef][Medline] [Order article via Infotrieve] |
24. | Hirano, T., Ishihara, K., and Hibi, M. (2000) Oncogene 19, 2548-2556[CrossRef][Medline] [Order article via Infotrieve] |
25. | Hirota, H., Yoshida, K., Kishimoto, T., and Taga, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4862-4866[Abstract] |
26. |
Kunisada, K.,
Tone, E.,
Fujio, Y.,
Matsui, H.,
Yamauchi-Takihara, K.,
and Kishimoto, T.
(1998)
Circulation
98,
346-352 |
27. | Yamauchi-Takihara, K., and Kishimoto, T. (2000) Int. J. Exp. Pathol. 81, 1-16[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Mascareno, E.,
El-Shafei, M.,
Maulik, N.,
Sato, M.,
Guo, Y.,
Das, D. K.,
and Siddiqui, M. A. Q.
(2001)
Circulation
104,
325-329 |
29. | Omura, T., Yoshiyama, M., Ishikura, F., Kobayashi, H., Takeuchi, K., Beppu, S., and Yoshikawa, J. (2001) J. Mol. Cell. Cardiol. 33, 307-316[CrossRef][Medline] [Order article via Infotrieve] |
30. | Negoro, S., Kunisada, K., Tone, E., Funamoto, M., Oh, H., Kishimoto, T., and Yamauchi-Takihara, K. (2000) Cardiovasc. Res. 47, 797-805[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Kunisada, K.,
Hirota, H.,
Fujio, Y.,
Matsui, H.,
Tani, Y.,
Yamauchi-Takihara, K.,
and Kishimoto, T.
(1996)
Circulation
94,
2626-2632 |
32. |
Kodama, H.,
Fukuda, K.,
Pan, J.,
Sano, M.,
Takahashi, T.,
Kato, T.,
Makino, S.,
Manabe, T.,
Murata, M.,
and Ogawa, S.
(2000)
Am. J. Physiol. Heart Circ. Physiol.
279,
H1635-H1644 |
33. |
Liu, S.,
and Schreur, K. D.
(1995)
Am. J. Physiol. Cell Physiol.
268,
C339-C349 |
34. | Liu, S. J., and McHowat, J. (1998) Am. J. Physiol. Heart Circ. Physiol. 44, H1462-H1472 |
35. |
Liu, S. J.,
Kennedy, R. H.,
Creer, M. H.,
and Howat, J.
(2003)
Am. J. Physiol. Cell Physiol.
284,
C826-C838 |
36. |
Bers, D. M.,
Li, L.,
Satoh, H.,
and McCall, E.
(1998)
Ann. N. Y. Acad. Sci.
853,
157-177 |
37. |
Bassani, J. W. M.,
Bassani, R. A.,
and Bers, D. M.
(1993)
Am. J. Physiol. Cell Physiol.
265,
C533-C540 |
38. |
Balligand, J.-L.,
Ungureanu-Longrois, D.,
Simmons, W. W.,
Pimental, D.,
Malinski, T. A.,
Kapturczak, M.,
Taha, Z.,
Lowenstein, C. J.,
Davidoff, A. J.,
Kelly, R. A.,
Smith, T. W.,
and Michel, T.
(1994)
J. Biol. Chem.
269,
27580-27588 |
39. | Yu, X.-W., Kennedy, R. H., and Liu, S. J. (2000) J. Mol. Cell. Cardiol. 32, A72 (Abstr. J10) |
40. | Wollert, K. C., and Drexler, H. (2001) Heart Fail. Rev 6, 95-103[CrossRef][Medline] [Order article via Infotrieve] |
41. | Murakami, M., Narazaki, M., Hibi, M., Yawata, H., Yasukawa, K., Hamaguchi, M., Taga, T., and Kishimoto, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11349-11353[Abstract] |
42. | Xie, Q. W., Whisnant, R., and Nathan, C. (1993) J. Exp. Med. 177, 1779-1784[Abstract] |
43. | Kleinert, H., Wallerath, T., Fritz, G., Ihrig-Biedert, I., Rodriguez-Pascual, F., Geller, D. A., and Förstermann, U. (1998) Br. J. Pharmacol. 125, 193-201[Abstract] |
44. |
Singh, K.,
Balligand, J. L.,
Fischer, T. A.,
Smith, T. W.,
and Kelly, R. A.
(1996)
J. Biol. Chem.
271,
1111-1117 |