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Introduction
Adrenoreceptors (AR) play essential roles in various
physiological and pathophysiological processes of the heart
by means of subtype-specific signaling. Current clinical
therapeutic strategies against this system are therefore an
important part of the management of many disorders, such as
coronary artery disease and chronic heart failure. While it is
known that signal transducers and activators of
transcription 3 (STAT3) as a cytoplasmic transcription factor mainly
mediates cytokine- and growth factor-directed transcription
under the stimulation of receptors, numerous data have
shown that myocardial STAT3 activity is required for
regulating various processes such as cardiac growth, function,
tissue architecture (neovascularization and ventricular
remodeling post-myocardial infarction), cellular
survival[1], apoptosis, and even providing protection against various
cardiovascular stresses (eg
ischemia)[2]. For instance, targeted the disruption of the mouse STAT3 gene is associated
with embryonic lethality[3]. Moreover, cardiomyocyte-restricted STAT3 knock-out mice exhibited greater
inflamma-tion, cardiac fibrosis, and heart failure with advanced
age[4]. Endogenous STAT3 has recently been shown to improve
cardiac function, increase vascularization, exhibit
anti-apoptotic effects, and regenerate cardiomyocytes following
stimulation with granulocyte colony stimulating factor in the
infarcted mouse heart[5]. Interestingly, accumulating evidence
suggests there is crosstalk between adrenoreceptors and
the STAT3 signaling pathways. Elucidating the potential
mechanisms of this crosstalk may provide new therapeutic
targets for heart disorders. This article reviews the current
understanding of these signaling pathways, and importantly
details mechanisms of potential crosstalk.
Overview of STAT3 signaling
Intense investigation into STAT transcript factors has
shown them to have important roles in tumor genesis,
inflammation, and homeostasis[6]. Seven mammalian STAT
proteins have been identified and designated as STAT1, 2, 3,
4, 5α, 5β, and 6. STAT3 ranges in size from 750 to 850 amino
acids, contains 6 conserved domains (an amino terminal
domain, a coiled-coil domain, a DNA-binding domain, a linker
domain, a Src homology (SH) 2 domain, and a transcriptional
activation domain), and exists in 3 natural isoforms
(STAT3α, STAT3β, and
STAT3γ). The C-terminal-truncated
STAT3β and STAT3γ behave as dominant_negative proteins which
functionally compete with their full-length counterparts to alter
or inhibit target gene expression. Several studies have
implicated STAT3 in regulating fundamental cellular biological
processes such as proliferation, differentiation, malignant
transformation, survival, and
apoptosis[7].
New insights into the STAT3 signaling pathway
The predominant receptors coupled to STAT3 signaling are the
interleukin-6 (IL-6) receptor family, which has a
gp130-associated signal transducer and interferon receptors. Although
these receptors do not possess tyrosine protein kinase
activity, their cytoplasmic domains contain binding sites of
tyrosine kinases. Additionally, mechanical stretch also
initiates the STAT3 pathway in rat cardiomyocytes.
Upon ligand binding, the receptors undergo a conformational change in
their cytoplasmic domain facilitating the recruitment of
Janus kinases (JAK), which confer tyrosine kinase activity.
JAK catalyze ligand-induced auto-phosphorylation and
phosphorylation of tyrosine residues on the receptor,
creating STAT3-docking sites, which leads to recruitment and
phosphorylation of tyrosine residues on STAT3. Moreover,
JAK also may generate a docking site for the SH2 domain
containing other signaling molecules, including protein
phosphatases and other adaptor proteins such as Shc, growth
factor receptor-binding protein 2 (Grb2), Cbl, the p85
subunit of phosphatidyl-inositol-3 kinase (PI3K), therefore
providing a platform for crosstalk between STAT3 signaling and
other signal transduction pathways.
Alternatively, many growth factor receptors such as
epidermal growth factor (EGF) can directly phosphorylate STAT3
through their intrinsic receptor tyrosine kinase activity.
Accumulating evidence indicates that G-protein-coupled
receptors (GPCR) (eg AR, angiotensin II receptors, and formyl
peptide receptor like-1) can also induce phosphorylation of
STAT3 at tyrosine residue 705 (STAT3-Tyr705) and serine
residue 727 (STAT3-Ser727)[8]. Notably, some non-receptor
tyrosine kinases such as the Src family (eg Src, Fyn, Lyn,
and Lck) may also directly activate STAT3 in the absence of
a classical stimulus[9]. In particular, there is evidence to
indicate that cooperation between Src family kinases and JAK is
required for full activation of STAT3, as inhibition of either
kinase family suppresses STAT
activity[10]. Also, Wen et
al demonstrated that Bmx, one of 7 members of Bruton's
tyrosine kinase (Btk)/Tec non-receptor tyrosine kinase family,
transactivated STAT-mediated gene expression in salivary
and lung epithelial cells[11]. Important advances have been
recently made with regard to the role of G proteins in
regulating STAT3 signaling. Indeed, Src and STAT3 are underlying
effectors of G proteins. In particular,
Gαo and Gα2, which belong to the
Gαi family of G proteins, induce transformation
of fibroblasts through a Src/STAT3 signaling pathway.
Phosphorylation of STAT3 facilitates homodimerization or
heterodimerization through reciprocal SH2-phosphotyrosine
interactions. STAT3 dimers are rapidly transported from the
cytoplasm to the nucleus, and are dependent on the
interactions of certain domains, such as coiled-coil and
DNA-binding domains, with the nuclear pore complex involving
importin-α3[12]. Cytoplasmic trafficking of STAT3 is an
active process and the phosphorylation of STAT3-Tyr705 is
not sufficient for the nuclear translocation of STAT3. STAT3
co-localizes with endocytic vesicles in transit from the cell
membrane to the perinuclear region, in response to growth
factor stimulation. The disruption of endocytosis, with
specific inhibitors, blocks STAT3 nuclear translocation and
STAT3-dependent gene regulation. These results suggest
that cytoplasmic transport of STAT3 may be dependent on
receptor-mediated endocytosis following receptor
activation[13]. Once in the nucleus, it regulates transcriptional activity of
target genes by binding to specific DNA response elements.
In contrast, 3 export signals have been defined in the
C-terminal part of the coiled-coil domain, the DNA binding
domain, and the linker domain (ie
STAT3306-318, STAT3404-414,
STAT3524-535), which play an important role in regulating
nuclear export of STAT3[14]. Recently, STAT3 has been shown
to bind to CREB-binding protein (CBP) and acetylated
histone H4 within the nucleus to form dot-like structures, termed
STAT3 nuclear bodies, that may be either directly involved
in active gene transcription or serve as reservoirs of
activated STAT3[15]. Furthermore, STAT3 may also interact with
other transcription factors to modulate target gene
transcrip-tion. For example, LIM homeodomain transcription factors
such as islet1 (Isl1), which is important for the differentiation
of motor neurons and organogenesis of the heart, interacts
with JAK and STAT3 in COS-1, HepG2, and a human
neuroblastoma cell line, SH-SY5Y, to form a complex. This
complex then activates JAK1 by phosphorylation, which in turn
increases tyrosine phosphorylation, facilitating STAT3
recruitment, leading to increases in DNA binding activity
and expression of target genes[16]. Other transcription
factors known to interact with STAT3 include c-Jun, Sp1, and
nuclear factor-κβ (NF-κβ)[17].
Recent studies indicate that the formation of STAT dimers
may occur independently of tyrosine phosphorylation and
that substantial levels of STAT proteins occur in the nucleus
of unstimulated cells. STAT3 may continuously shuttle
between the cytosol and the nucleus of unstimulated cells in a
manner independent of phosphorylation of Tyr705.
Further-more, confocal real time imaging and pulse fluorescence
localization after photobleaching technology indicated that
the decrease in STAT3 nuclear export contributed to the
nuclear accumulation of STAT3 following stimulation with
IL-6[18]. Increasing evidence indicates that
post-transcriptional modification may have an important role in the
activation of STAT3. Yuan et al recently reported that STAT3 was
also acetylated on lysine residue
685[19]. Correspondingly, unphosphorylated STAT3 (ie complete absence of tyrosine
phosphorylation) can also drive gene expression, such that
increases in STAT3 levels that follow its IL-6-dependent
activation by tyrosine phosphorylation are reported to drive a
distinct subset of genes. Furthermore, mRNA or proteins
whose expression is driven by overexpression of
unphos-phorylated STAT3 have been presented at high levels in
many cancers. Hence, STAT3 serves 2 quite distinct roles in
cytokine-dependent transcription: (i) as a part of the primary
response through the action of STAT3 dimers; and (ii) as a
secondary part of the complete response through the action
of increased amounts of unphosphorylated
STAT3[20].
The role of STAT3 on transcription is similar with STAT1,
which drives the constitutive expression of several genes in
the complete absence of tyrosine phosphorylation, a
function quite distinct from its role in inducible,
phosphorylation-dependent gene expression in response to interferons
and other cytokines. However, there is no data to indicate
that STAT3 is methylated despite several studies showing
that methylation of STAT1/6 is essential for their activation.
STAT3 is phosphorylated by many different kinases which
would appear to be dependent on species, cell type, and
eliciting stimulus. For example, Ser727 is phosphorylated by
several kinases, including Cdk5, H7-sensitive kinase,
protein Kinase Cd, zipper-interacting protein
kinase[21], PI3K, and mitogen-activated protein kinase (MAPK) cascade, that is,
MEK kinase 1, SEK-1/MKK-4, extracellular signal-regulated
kinase (ERK), p38, c-Jun NH2-terminal kinase (JNK),
ribosomal protein S6 kinases, mitogen- and stress-activated protein
kinase 1, transforming growth factor b-activated kinase
1[22], and Ca2+/calmodulin-dependent kinases. Indeed, signal
transduction from the plasma membrane to the nucleus by
STAT proteins is widely represented as exclusively a soluble
cytosolic process. Shah et al recently showed that the
endocytic pathway displayed important effects in IL-6/STAT3
signaling[23]. For example, IL-6 could enhance the
association of cytoplasmic phosphorylated STAT3-Tyr705 with the
purified early endosome fraction. Also, the inhibition of
endocytosis by transfection with dominant negative
amphi-physin A1, epsin 2a, dynamin K44A, and the clathrin light
chain, resulted in an inhibition of IL-6-stimulated STAT3
transcriptional function, indicating that endosome-mediated
trafficking of STAT3 may be required for optimal signal
trans-duction.
Downregulation of STAT3 signaling At least 8 different
mechanisms have been implicated in downregulating STAT3
signaling. However, the molecular basis of these has not
been well characterized. Several cytoplasmic tyrosine
phosphatases, including tyrosine phosphatases containing
a SH2 domain (SHP-1), CD45, and protein tyrosine
phosphatase 1B, are implicated in the dephosphorylation of
STAT3 signaling. In addition, suppressors of cytokine
signal proteins (SOCS) are potentially key negative regulatory
modulators of STAT3 signaling as they bind to receptor sites
and/or JAK catalytic sites. Moreover, the protein inhibitor
of activated STAT3 (PIAS3) was found to be specific for the
inhibition of activated STAT3[24]. Scaffolding proteins such
as the STAT3-interacting protein (StIP1), a novel protein
consisting of 12 WD40 repeats, can also inhibit STAT3
signaling by modulating the formation of multiprotein complexes
that are central in the regulation of signal transduction,
transcription, and targeted proteolysis. Collum
et al demonstrated that StIP1 had a high affinity for unphosphorylated
JAK and STAT3, and when overexpressed blocks STAT3
activation and dimerization/DNA binding, nuclear
translo-cation, and reporter gene transcription following stimulation
with IL-6[25].
Other proteins involved in the regulation of STAT3
include genes associated with retinoid-interferon-induced
mortality-19, which regulates nuclear translocation of
STAT3[26], and TEL/ETV6, a member of the ETS family of transcription
factors that represses STAT3 transcriptional activity
independent of TEL DNA binding[27]. A Ras homologue member
I (ARHI), a novel imprinted tumor suppressor gene, recently
was shown to form a complex with STAT3 in the cytoplasm
which resulted in the prevention of STAT3 accumulation in
the nucleus following stimulation with IL-6. ARHI markedly
reduced STAT3 binding to DNA and STAT3-dependent
promoter activity while only moderately affecting STAT3
phosphorylation[28]. In addition, the ubiquitin-proteasome
degradation pathway, which modulates the turnover of cytokine
receptors and activated JAK through various E3 ligase
com-plexes, may also serve as a highly specific mechanism to
inhibit STAT signaling[29]. Finally, receptor internalization
and secretion of soluble receptors have also been implicated
in the regulation of the JAK/STAT3 pathway.
In light of this, it is clear that the regulation of STAT3
signaling is a complex process involving a network of
partners including membrane receptors, interaction with
membrane-proximal region signaling molecules, modulation of
STAT3 through protein degradation/phosphorylation/acetylation, and crosstalk with the other signal transduction
pathways, in order to facilitate intra-nuclear coordination
with the other transcription factors.
Overview of AR signaling in the heart
As AR belong to GPCR family, they utilize various
G-protein subunits as part of their signaling cascade.
Cardio-myocytes express at least 6 AR subtypes, including
α1A, α1B, α1D,
β1, β2, and
β3. With respect to the classic pathway of
AR signaling, it is accepted that α-AR couple
Gq/phospholipase Cβ
(PLCβ)/inositol triphosphate
(IP3) and activate diacylglycerol/calcium and protein kinase C (PKC) signaling,
while β-AR are coupled to the Gs/i/adenylate
cyclase/cAMP/protein kinase A (PKA) pathway. On the other hand,
increasing evidence suggest novel mechanisms through which
AR can also induce responses by activating transduction
systems that do not involve G proteins through the
receptor_protein interaction. For example, Shenoy
et al recently demonstrated that β-arrestins mediated
β2-AR signaling to ERK1/2 independent of G-protein activation in HEK293
cells[30]. However, the significance of this novel signaling
mechanism in the cardiovascular system remains unclear.
α1-AR signaling
α1-AR signaling in the heart is diverse
and involves the activation of multiple signaling pathways.
It is well established that 3 subtypes of
α1-AR (α1A, α1B, and
α1D) mobilize intracellular
Ca2+ and activate PKC via
Gq/11. However, α1-AR have also been shown to activate pertussis
toxin-sensitive G proteins, such as Go,
and mediate the contractile response
in rat aorta. In addition, several lines of
evidence indicate that a1-AR are also coupled to alternative
effectors, such as phospholipase D, calcium/calmodulin
sensitive kinases, the
Na+/H+ exchanger,
Na+, K+-ATPase, and various ion currents including the L-type
Ca2+ current, the transient outward current, the delayed rectifier
K+ current, and the acetylcholine-activated
K+ current. Thus, α1-AR underpin many key processes in the heart including
myocardial contraction, growth response of
cardiomyocytes/fibroblasts, the cell cycle, differentiation and hypertrophy,
as well as survival and apoptosis[31]. The function of
α1-AR in the cardiovascular system has been extensively
studied[32]. Moreover, studies in recent years utilizing transgenic mice
overexpressing α1-AR, or with targeted disruption of genes
associated with α1-AR signaling in the myocardium, have
provided insights into mechanisms of α1-AR-induced
cardiac hypertrophy. Cardiac overexpression of
Gq in transgenic mice results in hypertrophy, decreased ventricular function,
and loss of a-AR inotropic responsiveness. In contrast,
upon overexpressing a carboxyl-terminal peptide of the
a subunit Gaq (a functional
Gq knockout), pressure overload induced myocardial hypertrophy was reduced by 60%_70%,
which implies that 30%_40% of the hypertrophic response
is independent of PLC/PKC[33]. O'Connell
et al recently reported that gene ablation of the
α1A and α1B subtypes in mice
resulted in a maladaptive form of reactive cardiac
hypertrophy from pressure overload, with a predisposition to heart
failure[34]. Given the potentially important role of
α1-AR signaling in the heart, further work is necessary to elucidate
crosstalk between this pathway and others involved in
cardiac hypertrophy signaling. Indeed, recent studies from our
laboratory have shown that filamin C can interact with 3
subtypes of α1-AR[35], while bone morphogenetic protein-1
can only specifically interact with α1A-AR. Another protein
that interacts with α1-AR is spinophilin[36].
β-AR signaling Within the heart, 3 subtypes of
β-AR have been cloned and identified pharmacologically. Recent
pharmacological studies have identified the existence of a
fourth subtype of β-AR in human and rat cardiac tissue,
which remains to be cloned[31]. β-AR are associated with
myocardial contraction, growth control, cell survival, and
apoptosis. Several β-AR subtypes can initiate multiple
intracellular signaling pathways in addition to Gs/adenylate
cyclase/PKA. For instance, recent studies demonstrate that
β2-AR are coupled to MAPK and cytoplasmic
phospholipase A2, via Gi in cardiomyocytes, which enhance cardiac
contraction and increase cardiomyocyte survival via
Gi/PI3K/Akt[37] and proliferation during early postnatal life. Wang
et al recently reported that in the PLC epsilon-deficient mouse,
cardiac contractility was attenuated in response to
isoprenaline and was independent of both β-AR-mediated cAMP
production and the size of the sarcoplasmic reticulum
calcium pool[38]. Furthermore, these knock-out mice were more
susceptible to the development of hypertrophy than wild
type mice. Hence, β-AR are believed to utilize different
signaling molecules to activate alternative compensatory
mechanisms under conditions of altered or defective classic
signaling.
Relatively little is known about the precise signaling
pathway associated with the stimulation of
β3-AR in the myo-cardium. Until
recently, β3-AR were purported to be
functional via the activation of adenyl cyclase and cAMP-dependent
phosphorylation. However, β3-AR do not possess a
C-terminal phosphorylation site for PKA or the b-AR kinase;
therefore, its signaling pathway may be different from that of
β1- and β2-AR. In human ventricular biopsies and
β3-AR knock-out mice, β3-AR was associated with reduced cardiac
contractility through Gi/o and production of nitric oxide via
endothelial constitutive nitric oxide
synthase[39]. In contrast, overexpression of
β3-AR in myocardium increases adenyl cyclase activity and enhances cardiac performance
Interes-tingly, it appears that a reciprocal relationship exists between
β3- and β1-AR in cardiomyocytes. Germack
et al recently observed that β3-AR were functionally upregulated and
coupled to the Gi protein in rat neonatal cardiomyocytes
following chronic exposure to noradrenaline, while
β1- and β2-AR were
downregulated[40]. This pattern is also observed in
human heart failure, as downregulation of
β1-AR is accompanied by a 2_3-fold upregulation of
β3-AR expression[41]. Similar to
α1-AR signaling, β-AR are differentially associated with
a variety of proteins other than G
proteins[42]; those which interact with
β1-AR include endophilin-1, PSD95, cyclic
nucleotide Ras guanine nucleotide exchange factor (associated
with Ras activation), and GAIP-interacting protein carboxyl
terminus. Likewise, β2-AR have been shown to interact with
proteins, such as the adaptor protein, Grb2, and a non-receptor tyrosine kinase, Src. In addition, β-AR signaling is
modulated through the coordinated actions of various
cytoplasmic enzymes including G-protein-coupled receptor
kinases, which phosphorylate and thus desensitize the
receptor, cyclic nucleotide phosphodiesterases which
hydrolyze cAMP, and phosphatases which dephosphorylate
phosphoproteins[31]. Differential expression patterns,
heterogeneity of tissue distribution, and intracellular
compartmentalization of these proteins may contribute to diversity
in downstream signaling of β-AR. Therefore, the possibility
exists for crosstalk among the various signaling pathways
and regulation of a multitude of biological processes.
Crosstalk between AR signaling and the STAT3
pathway
Numerous studies have investigated the role of STAT3
and AR (α1 and β) in cardiac remodeling/hypertrophy,
ischemic injury, and ischemic preconditioning in recent
decades. Interestingly, accumulating evidence indicates that
these signaling pathways undergo reciprocal
crosstalk[43]. For example, we have recently demonstrated that STAT3
plays a crucial role in mediating cardiac hypertrophy,
induced by α1-AR stimulation in neonatal rat cardiomyocytes
(unpublished data), although the underlying mechanisms are
not fully understood.
Intercellular crosstalk: interaction between
cardio-myocytes and non-cardiomyocytes Several studies show that
interaction between cardiomyocytes and
non-cardiomyo-cytes may play a role in mediating crosstalk between AR and
STAT3 signaling (Figure 1). We have previously
demonstrated that isoprenaline per se does not induce the
phosphorylation of STAT3-Tyr705 in mouse cultured ventricular
cardiomyocytes or fibroblasts. In contrast, in
vivo administration of isoprenaline (ip 15 mg/kg) caused delayed
phosphorylation of STAT3-Tyr705 in myocardium at 60 and 120
min, which was accompanied by an increase in myocardial
expression and serum levels of the IL-6 family (mainly IL-6);
this activation of STAT3 was abolished by phosphodiesterase
inhibition[44], suggesting that the delayed activation might
be mediated by cAMP. Interestingly, using anti-murine
IL-6-neutralizing antibody could significantly inhibit
isoprenaline-induced phosphorylation of STAT3-Tyr705, suggesting
that IL-6 might mediate isoprenaline-induced
STAT3 activa-tion. In vitro studies further show that the secretion
of members of the IL-6 cytokine family induced by isoprenaline was
from mice fibroblasts rather than
cardiomyocytes[44]. Moreover, evidence supporting a paracrine mechanism was
identified by Fredj et al, using an in
vitro co-culture of cardio-myocytes and fibroblast, where cardiomyocyte hypertrophy
was found to be dependent on the secretion of angiotension
II from fibroblasts[45]. More recently, our data demonstrate
that isoprenaline-induced secretion of IL-6 in cardiac
fibroblasts from mice is mainly mediated by the
α2-AR-Gs-AC-cAMP (but not PKA) signaling cascade, whereas inhibiting
the Gi/PI3K pathway significantly enhanced, while
pre-treatment with SB203580 (an effective blocker of p38-MAPK)
abolished, the induction of IL-6 by isoprenaline. These data
indicate that the mechanism mediating crosstalk between
these 2 pathways may centre on IL-6-mediated
phosphorylation of STAT3[46]. Indeed, it is clear that communication
between cardiomyocytes and fibroblasts, which are major
components of myocardium, appears to be more complex
than initially anticipated.
Intracellular crosstalk Sasaguri et
al were the first to report that
α1B-AR stimulation caused phosphorylation of
tyrosine residues in JAK2 and STAT1[47]. In particular, they
showed that in vascular smooth muscle cells,
phenylephrine-induced protein synthesis was dependent on STAT1
activation and could be inhibited by AG490, an inhibitor of
JAK2. Furthermore, Briest et al demonstrated that
intravenous administration of noradrenaline in rats caused
phosphorylation of STAT3-Tyr705 in cardiomyocytes, but not
non-myocytes, but was accompanied by an increase in IL-6 mRNA
levels in both cardiomyocytes and non-cardiomyocytes.
However, IL-6 and STAT3 levels were unchanged in
non-cardiomyocytes following noradrenaline infusion, despite
phosphorylation of STAT3-Try705 being downregulated,
which was in keeping with in vitro results. Systematic
analysis of NF-IL-6, IL-6, IL-6 receptor, and STAT3 mRNA
expression in myocardium following noradrenaline administration
supports the hypothesis that noradrenaline-induced cardiac
hypertrophy may involve sequential signaling via NF-IL-6,
IL-6, IL-6 receptors, and STAT3[48]. Similarly, our laboratory
has recently shown that in cultured rat cardiomyocytes
α1-AR stimulation causes phosphorylation of STAT3 at Ser727,
as early as 5 min and delayed phosphorylation of Try705.
Notably, cardiac hypertrophy in response to
α1-AR activation can be significantly relieved via AG490 or an inhibitory
peptide of STAT3 (unpublished data). However, the precise
mechanisms remain to be elucidated.
Mechanisms underlying AR-mediated activation of STAT3 signaling
Transactivation In recent years, increasing evidence
suggests that transactivation of receptor tyrosine kinase by
GPCR is a general phenomenon. Stimulation of many GPCR
by a diverse range of agonists including lysophosphatidic
acid, angiotensin II, endothelin, and bradykinin, are able to
activate receptor tyrosine kinase by
transactivation[49]. For example, lysophosphatidic acid induces the release of the
heparin-binding EGF by a metalloproteinase, thereby
resulting in the transactivation of EGF
receptors[50]. In addition, GPCR have also been shown to transactivate other receptor
tyrosine kinases, such as the vascular endothelium growth
factor, the platelet-derived growth factor, and the
insulin-like growth factor, resulting in the formation of receptor
tyrosine kinase/GPCR complexes that trigger a complex
sequence of intracellular signaling events which include STAT3
signaling.
AR are also capable of transactivating receptor tyrosine
kinases (Figure 1). For instance, in neonatal rat
cardiomyo-cytes, binding of phenylephrine to
α1-AR results in EGF receptor transactivation, in turn regulating transcription of
atrial natriuretic peptide by an ERK-mediated
pathway[51]. Similarly, β2-AR-mediated transactivation of the EGF
receptor induces proliferation of cardiac fibroblasts through a
P13K/ERK pathway[52]. Intriguingly, we recently demonstrated
that α1-AR-induced phosphorylation of STAT3_Tyr705 required transactivation of EGF receptors (unpublished
data). Although investigations of transactivation of GPCR and receptor tyrosine kinase have provided new insights into
elucidating the interactions between various signal
transduction pathways, the precise mechanisms remain unclear.
Furthermore, it is important to be able to differentiate
between a receptor-complex-mediated signaling pathway and
simple receptor tyrosine kinase-mediated signaling.
G proteins Recent reports indicate that
Gas and Gai, which are important subunits of G-protein-mediated
β-AR signaling, directly modulate Src kinase activity by binding to its
catalytic domain, which induces a conformational change
enhancing accessibility to its active
site[53]. These results suggest that other members of the Src tyrosine kinase family
may also be downstream effectors of G-protein signaling. A
Src/STAT3 pathway has been established in various cells
types. Previous studies indicate that
β2-AR stimulation is associated with phosphorylation of its C-terminal (Tyr350),
which exposes a binding site for the SH2 moiety of Src. The
resulting binding between β2-AR and Src leads to the
phosphorylation of Src and the activation of a G-protein-linked
receptor kinase 2[54], which in turn phosphorylates Ser/Thr
residues at the C-terminal of the β2-AR, prompting the
binding of β-arrestin and internalization of the receptor--a critical
step in the resensitization and recycling of the
receptor[55]. In addition to the Src/STAT3 pathway, several lines of evidence
show that Gαs and Gαi also participate in the activation of
MAPK signaling. Therefore, the possibility exists for
Gαs and Gαi to modulate STAT3 activation via a MAPK/STAT3
pathway (as discussed later). In addition, Gq
and Gi have also been shown to activate members of the Btk
family[56]. While Btk are associated with MAPK signaling, some
members of the family, such as Bmx can directly induce STAT
activation[11]. Although all 3
α1-AR subtypes are frequently coupled to
PLC-β activation, via Gq/11,
α1B-AR can interact with
Gα14 and Gα16, while
α1D-AR couple to Gα14 rather than
Gα16[57]. Ho et
al report that Gα14 is associated with both
α2- and β2-AR
signaling[58]. In HEK293 cells, STAT3 activation
is mediated through Gα14 and involves multiple intermediates
including PLC-β, calmodulin-dependent kinase II,
PKCα,ε, c-Src, JAK2/3, Ras/Rac1, Raf-1, and
ERK[59]. Likewise, Gα16 also activates STAT3 via a c-Src/JAK- and ERK-dependent
cascade[60]. However, at present there are no data available
to support the existence of Gα14 in the heart. In addition to
Gα, other G-protein subunits
(Gβ,γ) also modulate the activity of Btk through direct binding to their PH/TH
domain[61]. This raises the possibility that
Gβ,γ could also mediate Btk/STAT3
signaling.
cAMP/PKA pathway cAMP/PKA signaling is the
prototypical pathway associated with β-AR stimulation and has
been implicated in a vast array of cellular processes
including cell proliferation and contraction. However, the response
would appear to be dependent on cell type and nature of the
stimulus. New evidence indicates that increased cAMP
levels inhibit angiotensin II-mediated JAK/STAT3 activation
by a variety of mechanisms. Giasson et
al observed that in vascular smooth muscle cells, increased cAMP abolished
Tyk2 phosphorylation and inhibited JAK-attenuated protein
synthesis and vasoconstriction following exposure to
angiotensin II[62]. Interestingly, cAMP-elevating agents were
also shown to inhibit IL-6-induced STAT activation in
monocytes[63]. Together, these data establish a link for crosstalk
between cAMP and JAK/STAT3. At present, it is not known
how cAMP inhibits Tyk2 activation. However, it may
involve effects on PKA since pharmacological inhibition of
PKA abolishes the effects of cAMP. Tyk2 may be a
downstream target of PKA. Alternatively, PKA may directly or
indirectly activate protein tyrosine phosphatases, such as
SHP-1, resulting in the dephosphorylation of JAK. In
contrast, Park et al found that an increase in cAMP was
associated with the activation of STAT3, and inhibiting PKA
did not alter STAT3 activation in FRTL-5
thyrocytes[64]. The picture may be even more complex as the inhibition of PKC
also blocked cAMP-induced activation of STAT3. These
results suggest that the connection between the cAMP and
JAK/STAT3 pathway is diverse and dependent on both cell
type and species studied.
Small GTPase Small GTPases are a family of more than
100 monomeric G proteins which act as molecular switches
in signaling cascades and are modulated by guanine
nucleotide exchange factors. They can be divided into 6
subfa-milies, Ras, Rho, Rab, Arf, Ran, and Rad. Ras and Rho are
known to play important roles in gene regulation and cytoskeleton assembly, while the other subfamilies are
primarily involved in cytoplasmic transportation and assembly
of the microtubule spindle. Many studies indicate that small
GTPases are involved in the activation of STAT3. Simon
et al demonstrated that STAT3 formed a complex with activated
Rac1 (a member of the Rho subfamily), which increases Ser727
phosphorylation of STAT3. In support of this,
overexpres-sion of a dominant negative mutant of Rac1 inhibited
EGF-mediated activation of STAT3[65]. Pelletier
et al subsequently showed that Rac1 was necessary for angiotensin II-induced
activation of JAK and involved the generation of reactive
oxygen species via NADPH. Interestingly, this caused a
biphasic response with regard to phosphorylation of
STAT3-Tyr705. The initial phase associated with JAK activation
had a quick onset, reaching its peak within 3_6 min, before
returning to baseline by 15 min. In the second or delayed
phase, JAK activation occurred at 60 min and was
maintained for a further 60 min. As the delayed phase was
abrogated by actinomycin D (transcription inhibitor), it suggests
that the delayed activation phase is dependent on
de novo protein synthesis, possibly involving
IL-6[66]. Similarly, other members of the Rho subfamily, namely Rac and Cdc42, are
implicated in b2-AR-mediated activation of JNK in smooth
muscle cells. Furthermore, b-AR-induced apoptosis would
appear to involve the activation of Rac1 in rat
cardiomyo-cytes[67]. Although members of the Rho family have a role in
a1-AR-mediated myocardial hypertrophy, it remains to be
established whether small GTPases, such as Rho, link AR to
STAT3 signaling in the heart.
Scaffold and adaptor proteins Scaffolding and adaptor
proteins are emerging as "bridges" which organize the
spatial orientation of signal transduction pathways within
mammalian cells. For example, growth factor receptor-binding
protein 2 (Grb2)-associated binder-1 (Gab1), one of 3
members of the Gab scaffolding protein family, interacts with
various signal molecules associated with the growth factor
receptor, the cytokine receptor, and GPCR signaling, such
as protein tyrosine phosphatase SHP2, the p85 subunit of
PI3K, PLCγ, and Grb2. In cultured rat cardiomyocytes,
stimulation with leukemia inhibitory factor (LIF) induces
phosphorylation of Gab1, and initiates its interaction with SHP2
and p85. This Gab1-SHP2 interaction is crucial in mediating
gp130-dependent cytokine-induced hypertrophy in
cardio-myocytes and involves the activation of
ERK5[68]. Interes-tingly, transient expression of Grb2 inhibits EGF-mediated
STAT3 activation, while silencing Grb2 with RNA
interference enhances STAT3
activation[69]. In addition, the direct
binding of Grb2 to β2-AR mediates receptor internalization
following stimulation with insulin[70]. Due to the number of
reports documenting the interaction between scaffold
proteins/adaptor proteins and AR or JAK/STAT3 signaling, it is
not unreasonable to suggest that they may mediate crosstalk
between AR signaling and STAT3 signaling through the
formation of the signal molecules complex.
PKC PKC is generally activated in response to
stimulation of GPCR and protein tyrosine kinase receptors, or via a
non-receptor tyrosine kinase mechanism. It has been
implicated in the regulation of cell differentiation, proliferation,
apoptosis, and growth response. At least 11 isoforms of
PKC have been identified and are classified into 3 major
categories based on differential requirements for
Ca2+ and lipids. Their downstream targets include the Rho kinase, ERK,
calcium/calmodulin-dependent protein kinase II, and several
ion channels. There is a considerable body of evidence to
indicate that PKC participates in the activation of STAT3 in
various cells, such as FRTL-5 thyrocytes, HepG2 cells, and
monocytes, in response to receptor stimulation or
mechanical stretch. For example, Schuringa et
al demonstrated that in unstimulated
HepG2 cells, PKCδ strongly associated with
SEK-1/MKK-4 and was released from this complex in an
IL-6-dependent manner to phosphorylate STAT3-Ser727, but not
STAT3-Tyr705[71]. This occurs independently of JNK-1.
Rottlerin, a PKCδ inhibitor, causes a dose-dependent
reduction in STAT3 transactivation and is coupled to a decrease
in IL-6-induced STAT3-Ser727 phosphorylation;
IL-6-induced STAT3-Tyr705 phosphorylation is unaffected.
Similarly, overexpression of dominant negative
PKCδ also reduces IL-6-induced phosphorylation of STAT3-Ser727, but
not STAT3-Tyr705. Several recent reports have shown a
direct association between PKC and STAT in the heart. Wang
et al demonstrated that PKC phosphorylation enhanced
transcription factor GATA-4 DNA binding activity and STAT1
interacted with GATA-4 to synergistically activate
angiotensin II and growth factor-inducible
promoters[72]. Xuan et al reported that ischemic preconditioning was associated
with simultaneous activation of STAT1 and STAT3 via 2
parallel signal transduction pathways, JAK1/2/STAT and a
PKCε/Raf-1/MEK1/2/ERK
cascade[73]. Our recent studies indicate that chelerythrine, an inhibitor of PKC, can inhibit the
delayed phosphorylation of STAT3 induced by
α1-AR stimulation in rat cardiomyocytes. Consistently, PMA, via the
activation of PKC, also can evoke the phosphorylation of
STAT3-Tyr705 (unpublished data). However, it is unclear
which subtype of PKC is responsible for the induction of
STAT3 activation by α1-AR. In addition, it is noteworthy
that PKC can phosphorylate its substrates on serine residues,
thus, per se may not directly activate STAT3 by tyrosine
phosphorylation. Novotny-Diermayr et
al provided some evidence to partly explain this
puzzle[74]. As the catalytic domain of
PKCδ interacts with the SH2 domain and part of
the adjacent C-terminal transactivation domain of STAT3,
this interaction does not seem to depend upon phosphotyrosine
SH2-mediated binding. However, it significantly enhances
the interaction of STAT3 and the IL-6 receptor subunit, gp130,
facilitating STAT3 phosphorylation by gp130 receptor
stimulation.
Cytoplasmic protein tyrosine kinase As mentioned
earlier, STAT3 is also activated by certain
non-receptor tyrosine kinases other than JAK. In particular, Src plays an
important role in mediating STAT3 signaling. Src
phosphorylates the adapter protein Shc, which in turn activates the
Ras/Raf/ERK signaling cascade. It has been demonstrated
that IL-3 stimulates c-Src kinase activity, which facilitates
the binding of c-Src to STAT3, leading to the
phosphorylation of STAT3 and its translocation to the nucleus. While a
dominant negative mutant of JAK2 had no effect on
IL-3-mediated activation of STAT3, a dominant negative mutant
of c-Src abrogated STAT3 activation and inhibited
IL-3-mediated myeloid cell proliferation. These results indicate that
JAK and STAT phosphorylation events are mediated by 2
independent pathways[75]. Interestingly, many GPCR are able
to increase the activity of Src tyrosine kinases through an
ill-defined mechanism. Furthermore, various
G-protein-mediated physiological functions are sensitive to tyrosine kinase
inhibitors. In Src-deficient mouse embryonic fibroblasts, the
internalization of β2-AR is prevented; however, they are still
coupled to MAPK signaling[76]. It has been shown that
Gαs and Gαi directly stimulate the kinase activity of
downregul-ated Src and modulate Hck, another member of the Src family
of tyrosine kinases, in a similar manner. There is evidence
that small GTPases, such as Rap1 or Ral, may be involved in
the mechanism by which Gαo activates Src. G proteins may
also activate signaling molecules, such as Rap1, p38, and
JNK, which go on to form a complex with Src resulting in the
activation of Src. These results would indicate that direct
modulation of Src kinases by G proteins may be a common
phenomenon.
AR are associated with the activation of Src in various
cells, including airway smooth muscle cells. For instance,
β2-AR stimulation initiates the assembly of a protein complex
containing the receptor-activated c-Src and
β-arrestin[55]. Furthermore, it is generally accepted that Ras-dependent
activation of MAPK by GPCR, requires the activation of the
Src family tyrosine kinases. In cardiomyocytes, a
Gi/Gβγ, Src, Ras/Raf-1, and ERK cascade mediate isoprenaline-induced
myocardial hypertrophy[77]. In cardiac fibroblasts,
pre-incubation with PP2 (Src inhibitor) decreases
isoprenaline-mediated phosphorylation of EGF receptors and ERK activa-tion[52]. In PC12 cells stably expressing
α1-AR, noradrenaline stimulates Src tyrosine phosphorylation, while PP2
completely blocked ERK activation and cell
differentiation[78]. However, it remains to be established if there is a direct
association among AR/Src/STAT3 in the heart.
MAPK MAPK represent a family of serine/threonine
protein kinases and at least include
ERK1/2, p38, JNK, and ERK5. They are involved in the activation of other protein
kinases and transcription factors. MAPK share many
upstream and downstream kinases and transcription factors
that interact and integrate within these pathways. Several
studies have indicated that GPCR activate MAPK cascades
by multiple pathways, which include signaling molecules
such as Ras, Raf, PKC, Ca2+, and even tyrosine
kinase-dependent and independent pathways. AR-induced MAPK
signaling has been intensively studied in various species
and cell types (Figure 1). However, crosstalk between MAPK
and JAK/STAT3 signaling would appear to be more complex
and dependent on the species studied, cell type and nature
of the stimulus. For example, IL-6 activates the Ras/MAPK
cascade via JAK2, but MAPK also stimulate JAK
phosphorylation[79].
Kunisada et al demonstrated that LIF caused tyrosine
phosphorylation of gp130, JAK1, and
STAT3[80]. They also demonstrated that the activation of gp130 could trigger, in
parallel, both a MAPK cascade and STAT3 signaling in rat
cardiomyocytes. IL-1β and LIF are potent stimuli which can
cause cardiac hypertrophy in rat. Data indicate that
IL-1b-induced phosphorylation of STAT3-Tyr705 occurred
relatively late at 60 min, compared with that induced by LIF
within 10 min. Interestingly, they were both associated with
ERK activation. Pharmacological inhibition of ERK and p38
abolished the delayed phosphorylation of STAT3 and reduced atrial natriuretic factor expression by 70% following
exposure to IL-1β[81]. In addition, angiotensin II induced
rapid phosphorylation of STAT3-Ser727 in rat
cardiomyo-cytes, which was accompanied by an initial
dephosphorylation of STAT3-Tyr705 in the first 30 min and
phosphorylation of STAT3-Tyr705 after 90 min. All of these effects were
abolished by the ERK inhibitor,
PD98059[82]. These data suggest an important role for ERK and p38 in the delayed
activation of STAT3 induced by angiotensin II.
Nevertheless, we recently demonstrated that, compared with JNK, ERK
and p38 do not participate in α1-AR-mediated
phosphorylation of STAT3-Tyr705. In contrast, the
Gq/PLC/ERK pathway is important in the phosphorylation of
STAT3-Ser727 induced by α1-AR stimulation in neonatal rat cardiomyocytes
(unpublished data).
Interestingly, ERK-induced phosphorylation of
STAT3-Ser727 is sometimes associated with the inhibition of
STAT3-Tyr705 phosphorylation. Previously, studies in bone
marrow cells, hepatocytes, and HEK293 cells indicated that
MEK/ERK activation could rapidly (within 5 min) inhibit STAT3
tyrosine phosphorylation and DNA-binding activity
following exposure to IL-6[83]. In contrast to this, phenylephrine
inhibited IL-6-induced phosphorylation of STAT3, in primary
culture hepatocyte and HepG2 cells, which overexpress
α1B-AR via an ERK-dependent mechanism. This inhibition was
relatively late (occurring 2 h post stimulation) and was
abolished by either a tyrosine phosphatase blocker or MEK
inhibitor, but not a JAK1/2
inhibitor[84]. However, this inhibitory mechanism requires further exploration. In contrast,
recent data show Ser727 phosphorylation is required to
achieve maximal transcriptional activity of STAT3. For
example, Schuringa et al found that IL-6-induced
transactiva-tion of STAT3 and Ser727 phosphorylation was
independent of ERK-1 or JNK activity, but involves a signaling pathway that
includes Vav, Rac-1, MEK kinase, and
SEK-1/MKK-4[85]. The mechanism underlying the enhanced transcriptional activity
of STAT3-Ser727 might involve the selective recruitment of
co-activators, such as p300. While the AR, MAPK, and
JAK/STAT3 signaling pathway are 3 important mechanisms
mediating cardiac hypertrophy, elucidation of their interaction
will provide new insights for the development of novel
clinical strategies for this disorder.
Acetylation of STAT3 It is generally accepted that the
phosphorylation of STAT3-Tyr705 plays a critical role in
dimerization and induction of gene transcription, and that
post-translational modification of Ser727 modulates STAT3
functionality. Nevertheless, accumulating evidence indicates
that unphosphorylated or tyrosine-mutated STAT proteins
can still form dimers and induce gene
transcription[86]. Therefore, another type of regulation must contribute to the
stable dimerization of STAT. Recently, Wang et
al showed that STAT3 could be acetylated in the C-terminal
transcriptional activation domain at lysine 685 by its co-activator,
p300/CBP, both in vivo and in
vitro[87]. This modification enhances its sequence-specific DNA binding ability and
transactivation activity. In contrast, histone deacetylase
(HDAC) acts as a negative modulator. Around the same
time, Yuan et al[19] also reported the same post-translational
modification in other cells following cytokine stimulation. In
PC3 cells co-transfected with p300 and a series of STAT3
mutants (Tyr705, Ser727, and Arg585), STAT3
was still acetylated, indicating that acetylation occurs independently
of phosphorylation and SH2 domain activity. Moreover, the
acetylation of Lys685 plays a critical role in STAT3
dimeriza-tion, transcription of cell growth-related genes, and
modulation of the cell cycle in response to cytokines. Although
STAT3 deacetylation is mediated by type 1 HDAC (mainly
HDAC3), there is still controversy over which lysine
residues in STAT3 are acetylated. Based on previous studies
showing that the IL-6/JAK/STAT3 pathway is involved in
the transcriptional modulation of human angiotensinogen
(hAGT) in hepatocytes, Ray et al recently demonstrated that
STAT3 was acetylated at its NH2 terminus on lysine
residues 49 and 87[88]. A double-point mutation of STAT3 at
these 2 conserved sites caused a 3-fold decrease of
IL-6-induced hAGT transactivation. Furthermore, binding of
HDAC1 and 4 (particularly HDAC1) to activate STAT3 acts
as an intra-nuclear molecular switch. This controls the
duration of the STAT3 transcriptional response and contributes
to the replenishment of cytoplasmic pools depleted of latent
STAT3. Therefore, further work is required to investigate
the cell-type-specific differences in acetylation sites of
STAT3. A considerable body of data indicates that
activation of ERK is a key factor in mediating
phenylephrine-induced cardiac hypertrophy. Interestingly,
phenylephrine could increase the transcriptional activation ability of CBP
and p300, as this effect is dependent upon its ability to
activate ERK signaling[89]. More importantly, the inhibition of
CBP and p300 decreased atrial natriuretic factor gene
expression following exposure to phenylephrine. Based on this
and the fact that CBP and p300 modulate STAT3 acetylation,
whether it is possible that a relationship exists between
AR/ERK/CBP/p300 and STAT3 remains to be established along
with its biological significance.
Summary
There is mounting evidence of the crucial roles of AR
and STAT3 signaling pathways in various heart
pathophysiological processes. Data suggest that the complex crosstalk
between the 2 signaling pathways appears to depend on
species/tissue type, development status, and stimulus.
Elucidation of the potential mechanisms of their crosstalk,
occurring at multiple cascade levels by different manners, will
provide new targets for the development of novel clinical
strategies for heart disorders.
Acknowledgements
We thank Dr James P SPIERS and Dr Elizabeth J KELSO
for valuable revision on the manuscript.
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