© 1999 American Heart Association, Inc.
Molecular Medicine |
Characterization of Insulin-Like Growth Factor-1–Induced Activation of the JAK/STAT Pathway in Rat Cardiomyocytes
From the Cardiopulmonary Division, Department of Internal Medicine, Keio University, Tokyo, Japan.
Correspondence to Keiichi Fukuda, MD, PhD, Cardiopulmonary Division, Department of Internal Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail kfukuda{at}mc.med.keio.ac.jp
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Abstract |
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Abstract—This study was designed to investigate whether insulin-like growth factor-1 (IGF-1) transduces signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and to assess the upstream signals of serine and tyrosine phosphorylation of STAT family proteins. Primary cultured neonatal rat cardiomyocytes were stimulated with IGF-1 (10-8 mol/L). JAK1, but not JAK2 or Tyk2, was phosphorylated by IGF-1 as early as 2 minutes and peaked at 5 minutes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. Tyrosine phosphorylation of STAT1 peaked at 15 minutes and correlated with that of JAK1, whereas that of STAT3 was sustained up to 120 minutes and was dissociated from the activation of JAK1. Tyrosine phosphorylation of STAT3 was unaffected by the preincubation with CV11974 (AT1 blocker), TAK044 (endothelin-1 receptor blocker), RX435 (anti-gp130 blocking antibody), PD98058, wortmannin, EDTA, or KN62 but was significantly attenuated by BAPTA-AM and chelerythrine. The time course of a gel mobility shift of SIE (sis-inducing element) coincided with the phosphorylation of STAT3. Serine phosphorylation of STAT1 peaked at 30 minutes and that of STAT3 was observed from 5 to 60 minutes. These results indicated that (1) IGF-1 activated JAK1 but not JAK2 or Tyk2 in rat cardiomyocytes; (2) IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3; and (3) the tyrosine phosphorylation of STAT3 was not caused by JAK1 alone, and protein kinase C and intracellular Ca2+ were required for phosphorylation.
Key Words: insulin-like growth factor-1 • JAK • STAT • cardiomyocyte • signal transduction
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Introduction |
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Insulin-like growth factor-1 (IGF-1) is a critical growth factor for various types of cells. Challenge of target cells with IGF-1 results in a pleiotropic response that can include cellular proliferation or differentiation, stimulation of amino acid uptake, glycogen metabolism, and induction of mRNA and protein synthesis. IGF-1 is also known to play important roles in cardiac hypertrophy and heart failure. Ito et al1 demonstrated that IGF-1 caused cardiac hypertrophy with enhanced expression of muscle-specific genes in cultured cardiomyocytes. Duerr et al2 reported that IGF-1 enhanced ventricular hypertrophy and function in in vivo rat heart with experimental myocardial infarction. Ambler et al3 reported that IGF-1 also improved cardiac dysfunction in doxorubicin-induced cardiomyopathy. Many recent studies have shown that IGF-1 increased [Ca2+] transient in cardiomyocytes, improved cardiac contractility,4 5 preserved ischemic myocardium from reperfusion injury,6 and protected against cardiac necrosis and inhibition of reperfusion-induced apoptosis of cardiac myocytes.7 Reiss et al8 reported that IGF-1 and the IGF-1 receptor system were upregulated in experimental myocardial infarction. Recent clinical studies have demonstrated that growth hormone improved heart failure in patients with dilated cardiomyopathy.9 IGF-1, previously known as somatomedin, increased with growth hormone treatment10 and is considered to play a critical role in improving cardiac function.2 These findings indicated that IGF-1 might be a critical growth factor for cardiomyocytes; however, its precise signal transduction pathways and their roles in cardiomyocytes remain unclear.11 12
The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathways are activated by various cytokines and growth factors such as interleukin-6 (IL-6), epidermal growth factor, and platelet-derived growth factor.13 We and others have recently reported that cardiotrophin-1 and leukemia inhibitory factor (LIF) caused cardiac hypertrophy and activated the JAK/STAT pathway in cardiomyocytes.14 15 16 We also reported that the JAK/STAT pathway was activated by angiotensin II in cultured cardiomyocytes17 and pressure-overloaded rat heart.18 Recently, Kunisada et al19 reported that overexpression of STAT3 using an adenovirus system augmented c-fos and atrial natriuretic factor (ANF) mRNA expression and protein synthesis in LIF-stimulated cardiomyocytes, whereas overexpression of dominant-negative STAT3 attenuated c-fos and ANF mRNA expression and protein synthesis. These results indicated that STAT3-dependent signaling might promote cardiomyocyte hypertrophy. Fujio et al20 reported that LIF phosphorylated STAT1 and induced the antiapoptotic cytoprotective gene bcl-xL, and that the activation of STAT1, but not STAT3, could induce bcl-xL in cardiomyocytes. Taken together, the JAK/STAT pathway might be critically involved in mediating cardiac hypertrophy and presenting a cytoprotective effect in cardiomyocytes.
IGF-1 activates various signaling pathways including the IRS-1/PI-3K, ras/raf-1/extracellular responsive kinase (ERK), and PTP1D pathways.21 However, it remains unclear whether IGF-1 can signal through the JAK/STAT pathway, especially in cardiomyocytes. Considering that IGF-1 has both hypertrophic and antiapoptotic effects on cardiomyocytes, and that other tyrosine kinase–binding receptors use this pathway, there is a possibility that IGF-1 signals through this pathway. In the present study, we report that IGF-1 induced characteristic activation of JAK1, STAT1, and STAT3 in rat cardiomyocytes and have assessed the upstream signals of serine and tyrosine phosphorylation of STAT1 and STAT3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Recombinant IGF-1 was provided by Fujisawa Pharmaceuticals Co Ltd. Antibodies to JAK1, JAK2, Tyk2, STAT1
/ß, STAT3, and ERK1/2,
specific substrate for Raf-1 (PLARTLSVAGLPGKK), and the synthetic
oligonucleotide probes for SIE and mutant SIE were
purchased from Santa Cruz Biotechnology. The sequences of the probes
have been described (SIE: 5'-GTGCATTTCCCG-TAAATCTTGTCTACA-3', mutant
SIE: 5'-GTGCATCCACCGT- AAATCTTGTCTACA-3'). Monoclonal antibody to
phosphotyrosine (4G10) was purchased from Upstate Biotechnology.
Polyclonal and monoclonal antibodies to phosphoserine were purchased
from Zymed Laboratories and Sigma. Polyclonal antiphosphospecific
mitogen-activated protein kinase (MAPK) antibody was purchased
from New England Biolabs.
Cell Culture
Primary cultures of cardiomyocytes were
prepared from the ventricles of 1-day-old Wistar rats as described
previously (Japan CLEA, Tokyo, Japan).14 The cells were
incubated in serum-free medium for 24 hours and then stimulated with
IGF-1 (10-8 mol/L).
Immunoprecipitation (IP) and Western Blot Analysis
The cells were lysed in a buffer as described
previously.17 The lysates were precleared by incubation
with protein A–Sepharose beads (Sigma). Precipitating antibodies were
added to the lysates, and the lysates were incubated for 3 hours at
4°C. Immunoprecipitates were pelleted with protein A–Sepharose beads
and washed 4 times with the lysis buffer. The precipitated proteins
were subjected to SDS-PAGE, and Western blot analysis was
performed as described.17
Kinase Activity Assays for Raf-1
Cells were lysed in the same buffer as above except that it did
not contain 10% glycerol, 0.1% SDS, or 1.0% deoxycholic acid. Raf-1
proteins were immunoprecipitated. The immunoprecipitates were incubated
with specific substrates (2.5 nmol/L) in the presence of 25 mmol/L
Tris-HCl (pH 7.4), 10 mmol/L MgCl2, 1
mmol/L DTT, 40 µmol/L ATP, 2 µCi of
[
-32P]-ATP, and 0.5 mmol/L EGTA. After
20 minutes of incubation at 25°C, aliquots (15 µL) were spotted on
P81 paper. The papers were washed five times for 10 minutes each in
0.75% phosphoric acid, dried, and counted by the Cerenkov
technique.
Gel Mobility Shift Assay
Nuclear extracts were prepared according to standard methods as
described previously.22 Briefly, harvested cells were
resuspended in 5 vol of hypotonic buffer, incubated for 10 minutes on
ice, and centrifuged. The cells were then resuspended in 2 vol
of the same buffer, Dounce-homogenized, and spun at
1000g for 10 minutes, and the pelleted nuclei were incubated
for 30 minutes at 4°C in high salt buffer. The nuclear extracts were
dialyzed against dialysis buffer overnight. The protein concentrations
were determined by Bradford’s method. Nuclear extracts (5 µg) were
incubated with 1 µg of poly(dI-dC)-poly(dI-dC) in 20 µL of 25
mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 0.5 mmol/L EDTA, and
10% glycerol for 20 minutes at 25°C. The samples were incubated with
1 fmol/L of radiolabeled probes (
5000 cpm) for 10 minutes at 25°C.
Binding reactions were resolved by 4% native polyacrylamide
gel electrophoresis containing 1x Tris-acetate/EDTA electrophoresis
buffer and visualized by autoradiography.
Statistical Analysis
Values are presented as mean±SD. Statistical
significance among mean values was evaluated with ANOVA. Student’s
t test was used when two values were compared. Differences
were considered to be significant at values of P<0.05.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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IGF-1 Phosphorylates JAK1 but Not JAK2 or Tyk2 in Cardiomyocytes
To determine whether IGF-1 transduces signaling through the JAK/STAT pathway in cardiomyocytes, we performed IP Western blotting to detect the tyrosine phosphorylation of JAK family kinases. Four members have been identified so far: JAK-1, JAK-2, JAK-3, and Tyk-2. JAK-1, JAK-2, and Tyk-2 are ubiquitous, whereas JAK-3 is expressed only in T lymphocytes. LIF was used as a positive control. IGF-1 phosphorylated JAK1 as early as 2 minutes, peaked at 5 minutes, and decreased thereafter (Figure 1A
). The time course of the
phosphorylation of JAK1 was equivalent to that of LIF.
In contrast, neither JAK2 nor Tyk2 was phosphorylated
after IGF-1 treatment (Figure 1B), whereas LIF clearly led to
phosphorylation of JAK2 and Tyk2. Similar results were
obtained in 3 additional experiments. These findings indicated that
JAK1, but not JAK2 or Tyk2, was involved in IGF-1 signaling.
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IGF-1 Activates the Raf-1/MAPK/ERK Kinase (MEK)/ERK Pathway
in Cardiomyocytes
IGF-1 activates Raf-1/MEK/ERK pathway in various types of
cells.23 A recent study suggested that this pathway plays
an important role in the serine phosphorylation of STAT
families. Thus, to confirm that IGF-1 activated this pathway in
cardiomyocytes, we detected the MAPKKK activity of Raf-1
and the phosphorylation of ERK after IGF-1 stimulation.
The MAPKKK activity of Raf-1 increased as early as 2 minutes, peaked at
5 minutes, and returned to the control level by 30 minutes (Figure 2A). The serine
phosphorylation of ERK1/2 was detected by Western blot
analysis using antiphosphospecific MAPK antibodies.
Phosphorylation of ERK1 and ERK2 increased at 2
minutes, peaked at 5 minutes, and decreased to the control level at 30
minutes (Figure 2B). These findings show that the Raf-1/MEK/ERK
pathway was also involved in IGF-1–regulated signaling in
cardiomyocytes.
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Tyrosine Phosphorylation of STAT1 and STAT3 by
IGF-1 in Cardiomyocytes
Next, we performed IP Western blot analysis to detect the
tyrosine phosphorylation of STAT families. STAT1 and
STAT3 were unphosphorylated in unstimulated cells.
After IGF-1 stimulation, tyrosine phosphorylation of
STAT1 was observed at 2 minutes and peaked at 15 minutes but could not
be observed at 30 minutes (Figure 3A).
Tyrosine phosphorylation of STAT1 by LIF was observed
at 5 minutes, peaked at 15 minutes, and returned to the control level
at 30 minutes. The time course of the phosphorylation
of STAT1 by IGF-1 was almost similar to that by LIF.
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In contrast, IGF-1–induced tyrosine phosphorylation of
STAT3 showed a pattern quite distinctive from that of STAT1. In this
unique time course, phosphorylation was observed as
early as 2 minutes, gradually increased to peak at 60 minutes, and was
still sustained at 120 minutes (Figure 3B
). The results were
reproducible in 5 separate experiments. Phosphorylation
of STAT3 by LIF was observed at 2 minutes, was maximal at 5 minutes,
and was not observed at 30 minutes. The time course of the
phosphorylation of STAT3 by IGF-1 was quite different
from that by LIF. The early phase of tyrosine
phosphorylation of STAT3 by IGF-1 could be explained by
JAK1, but it was difficult to explain that this slow time course of the
phosphorylation of STAT3 was caused by JAK1 alone,
suggesting that STAT3 was phosphorylated by another
kinase. Next, we compared the strength of IGF-1–induced
phosphorylation of STAT3 with that by LIF. We
stimulated the cells with various doses of LIF for 5 minutes or IGF-1
for 60 minutes and quantitated tyrosine phosphorylation
of STAT3 by densitometric analysis (Figure 3C). The
maximal strength of phosphorylation of STAT3 by IGF-1
was 12±3% of that by LIF.
Serine Phosphorylation of STAT1 and STAT3 by
IGF-1
Recent studies revealed that serine
phosphorylation of STAT1 and STAT3 was important for
maximal transcription.24 IP Western blotting was used to
determine whether IGF-1 induces serine phosphorylation
of STAT1 and STAT3 (Figure 4). Serine
phosphorylation of STAT1 by IGF was observed at 5
minutes and peaked at 30 minutes. Serine
phosphorylation of STAT3 increased at 5 minutes and was
sustained until 60 minutes. The time course of the serine
phosphorylation of STAT3 by IGF-1 was also slow, as was
the tyrosine phosphorylation.
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Gel Mobility Shift of SIE
The results of the IP Western blot analysis of STATs
indicated that STAT1 and STAT3 were differentially regulated in IGF-1
stimulation. Homodimers or heterodimers of STAT1 and STAT3 form
sis-inducing factor (SIF) complexes and translocate into the
nucleus. Therefore, a gel mobility shift assay was used to examine
sis-inducing element (SIE) (Figure 5). IGF-1 induced the formation of SIF
complex associated with intranuclear oligonucleotides
corresponding to SIE as early as 5 minutes. This DNA-protein
interaction was maximal at 60 minutes. The time course of the shift of
SIE bands corresponded to that of the tyrosine
phosphorylation of STAT3.
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Upstream Signals of the Tyrosine Phosphorylation
of STAT3
The time course of the tyrosine phosphorylation of
STAT3 was dissociated from the time course of the activation of JAK1.
To show whether IGF-1 induced secretion of other growth factors such as
angiotensin II, endothelin-1, or IL-6 family
cytokines, which may in turn phosphorylate STAT3 in
the late phase, we preincubated the cells with CV11974
(AT1 antagonist:
10-6 mol/L) or TAK044 (endothelin-1 type A and B
receptor antagonist: 10-6 mol/L) and
observed the tyrosine phosphorylation of STAT3 at 60
minutes (Figure 6A). CV11974 and TAK044
did not affect the tyrosine phosphorylation of STAT3,
suggesting that neither angiotensin II nor endothelin-1 was
involved in this phosphorylation. To confirm whether
paracrine-secreted IL-6 family cytokines are involved in
IGF-1–induced activation of STAT3, we preincubated the murine
cardiomyocytes with RX435 (anti-gp130 blocking antibody)
and stimulated the cells with either LIF or IGF-1 (Figure 6B).
Preincubation of RX435 strongly inhibited the
phosphorylation of STAT3 by LIF, whereas it did not
inhibit that by IGF-1, suggesting that paracrine-secreted IL-6 family
cytokines were not involved in this
phosphorylation.
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Next, we investigated whether other signaling pathways were involved in
the activation of STAT3 in IGF-1–mediated signaling. We preincubated
the cells with PD98059 (MEK inhibitor), wortmannin (PI-3K
inhibitor), BAPTA-AM, EDTA, KN62 (calmodulin
kinase II inhibitor), or chelerythrine (protein kinase C
[PKC] inhibitor) for 30 minutes and measured the
IGF-1–induced tyrosine phosphorylation of STAT3 at 60
minutes. The tyrosine phosphorylation of STAT3 was not
affected by PD98059, wortmannin, EDTA, or KN62 but was significantly
attenuated by BAPTA-AM and chelerythrine (Figure 7A). Densitometric analysis
confirmed that BAPTA-AM and chelerythrine inhibited the IGF-1–induced
tyrosine phosphorylation of STAT3 by 95% and 98%,
respectively (Figure 7B). These findings indicated that
intracellular Ca2+ and PKC were involved in
IGF-1–induced delayed tyrosine phosphorylation of
STAT3 in cardiomyocytes.
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Upstream Signals of the Serine Phosphorylation
of STAT1
Previous reports demonstrated that both raf-1/MEK/ERK-dependent
and -independent pathways were involved in serine
phosphorylation of STATs. Upstream signals of the
serine phosphorylation of STATs are now considered to
be both ligand and cell type specific. Thus, we preincubated the cells
with BAPTA-AM, EDTA, KN62, chelerythrine, PD98059, or wortmannin for 30
minutes and measured the IGF-1–induced tyrosine
phosphorylation of STAT1 (Figure 8). The serine
phosphorylation of STAT1 was not affected by any of the
inhibitors used. These results indicated that
IGF-1–induced serine phosphorylation STAT1 was
independent of the PKC, PI-3K,
Ca2+/calmodulin/CaMKII, and
raf-1/MEK/ERK pathways.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is increasing evidence for the involvement of the JAK/STAT pathway in insulin-mediated signaling, but the detailed interactions within this pathway or with other signaling pathways remain unclear. Moreover, it has not yet been determined how this pathway is involved in IGF-1–mediated signaling, although the insulin and IGF-1 receptors are closely related. In the present study, we found that IGF-1 activated JAK1 but not JAK2 or Tyk2 in cardiomyocytes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. The time course of the tyrosine phosphorylation of STAT1 was in accordance with that of JAK1 but that of STAT3 was sustained and was dissociated from the activation of JAK1. In addition, the time course of the mobility shift of SIE coincided with the phosphorylation of STAT3. Finally, PKC and intracellular Ca2+ were found to be required for the tyrosine phosphorylation of STAT3.
Giorgetti-Peraldi25 reported that in fibroblasts overexpressing insulin receptor both JAK1 and JAK2 were constitutively associated with Grb2 through the SH3 domains of Grb2, and that insulin induced the tyrosine phosphorylation of JAK1 but did not modify the tyrosine phosphorylation state of JAK2. However, Saad et al26 reported that insulin did induce tyrosine phosphorylation of JAK2 in insulin-sensitive tissues of the intact rat. Gual et al27 reported that in fibroblasts overexpressing insulin and IGF-1 receptor, insulin and IGF-1 lead to phosphorylation and activation of JAK1 and JAK2, and that JAK1 phosphorylated IRS-1 on sites different from those phosphorylated by the insulin receptor and interacted directly with phosphorylated insulin and IGF-1 receptor.27 We have observed that IGF-1 induced tyrosine phosphorylation of JAK1 but not JAK2 or Tyk2 in cardiomyocytes. Our findings for JAK1 in cardiomyocytes agree with the results obtained in fibroblasts overexpressing IGF-1 receptor27 ; however, we did not observe the enhancement of tyrosine phosphorylation of JAK2 in cardiomyocytes. One reason for this difference was the different cell types used. The IGF-1–induced tyrosine phosphorylation of JAK2 was observed in cells that overexpressed IGF-1 receptor, and the maximal phosphorylation of JAK2 was only 1.9±0.2-fold of the control. Thus, we believe that JAK1, but not JAK2, is critically involved in IGF-1–mediated signaling in primary cultured cardiomyocytes.
STAT proteins regulate cell growth and differentiation. Chuang et
al28 reported that insulin rapidly
tyrosine-phosphorylated STAT1 and increased its
specific binding activity to a GAS/ISRE consensus
oligonucleotide in HEP3B cells. They also reported that
JAK1, JAK2, and Tyk2 were not tyrosine-phosphorylated
at 15 minutes and concluded that this phosphorylation
was caused by a JAK-independent pathway. We have observed IGF-1
tyrosine-phosphorylated STAT1 in
cardiomyocytes. We could not conclude whether the upstream
signals of IGF-1–induced tyrosine phosphorylation of
STAT1 are JAK1 dependent or not. The time course of tyrosine
phosphorylation of JAK1 and STAT1 suggests that JAK1
mediated IGF-1–induced tyrosine phosphorylation of
STAT1 in cardiomyocytes. IGF-1–induced tyrosine
phosphorylation of JAK1 peaked at 5 minutes, decreased
at 15 minutes, and was apparently weaker than that induced by LIF.
According to the data presented by Gual et al,27
insulin-induced tyrosine phosphorylation of JAK1 peaked
at 2 to 5 minutes and decreased at 10 minutes. Thus, Chuang et
al28 may have missed the peak of JAK1
phosphorylation.
In contrast to STAT1, we observed that IGF-1 induced the sustained
tyrosine phosphorylation of STAT3 and mobility shift of
SIE in cardiomyocytes. Coffer et al29 reported
that the SIF complex (STAT3 dimer) was rapidly induced by insulin and
was sustained for several hours, and that ERK and PI-3K were not
required for this activation. Our observation of the IGF-1–induced
phosphorylation of STAT3 was in accordance with their
observations with insulin. It is possible to explain the early phase of
the activation of STAT3 by induction by JAK1, but it is difficult to
explain the entire time course of this phosphorylation
by JAK1 alone. The present study revealed that IGF-1–induced
tyrosine phosphorylation of STAT3 was blocked by
BAPTA-AM and chelerythrine but not by PD98059, wortmannin, EDTA, or
KN62. These findings indicated that this long-lasting
phosphorylation required certain levels of
[Ca2+]i and PKC but did
not require the MEK/ERK pathway, PI-3K, or
Ca2+/calmodulin kinase II. Recent
evidence indicated that the transforming tyrosine kinases encoded by
v-Src, v-Abl, Bmx, and v-Fps can induce STAT
activation,30 31 32 suggesting that their normal cellular
homologs such as c-Src and c-Fes may contribute to STAT activation
under physiological conditions.33
Moreover, a recent report revealed that insulin receptor-ß directly
phosphorylates STAT5, suggesting that STAT3 might be
another substrate for insulin and the IGF-1 receptor.34 35
Although we could not determine which tyrosine kinase was involved in
this phosphorylation, a
Ca2+-dependent PKC might exist upstream of the
kinase.
Recent studies have revealed that maximal activation of transcription
by STAT1 and STAT3 requires both tyrosine and serine
phosphorylation.24 Zhu et
al36 reported that interferon- induced both serine and
tyrosine phosphorylation of STAT1, and that STAT1
serine phosphorylation is more delayed than tyrosine
phosphorylation. They also reported that the ras/MAPK
pathway was not involved in interferon-–induced STAT1 serine
phosphorylation.36 The present study
revealed that the time course of the serine/tyrosine
phosphorylation of STAT1 by IGF-1 was similar to that
by interferon-. We have also shown that the upstream signal of
serine phosphorylation of STAT1 was independent of PKC,
PI3-K, Ca2+/calmodulin/CaMKII, or
raf-1/MEK/ERK pathways. These findings were also similar to those for
interferon-.
Chung et al37 reported that serine phosphorylation of STAT3 was mediated by both ERK-dependent and -independent pathways. Ceresa et al38 39 reported that insulin induced STAT3 serine phosphorylation by a Ras/Raf-1/MEK-dependent pathway. In the present study, we attempted to determine the upstream pathway of serine phosphorylation of STAT3 by IGF-1. The Raf-1/MEK/ERK pathway was partially involved in the serine phosphorylation of STAT3 (data not shown). However, we could not definitively determine the upstream pathway of the serine phosphorylation of STAT3. This is probably because the serine phosphorylation of STAT3 was sustained, and several pathways might be involved in this activation. The pathway upstream of the IGF-1–induced serine phosphorylation of STAT1 and STAT3 needs to be clarified in the future.
The mechanism of activation of the IGF-1–induced JAK/STAT pathway is both very complicated and quite different from other pathways induced by cytokines or other growth factors. Because STATs are critical mediators of cardiomyocyte function, precise analysis needs to be continued.
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Acknowledgments |
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This study was supported in part by research grants of the "Research for the Future" Program from the Japan Society for the Promotion of Science (JSPS-RFTF97I00201), research grants from the Ministry of Education, Science and Culture, Japan, and Health Science Research Grants for Advanced Medical Technology from the Ministry of Welfare, Japan. We wish to acknowledge Rie Inaba for technical assistance.
Received July 21, 1999; accepted August 31, 1999.
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