From the Departments of Pharmacology and Medicine,
College of Physicians and Surgeons, Columbia University, New York,
New York 10032 and ¶ Johnson and Johnson Pharmaceutical Research
and Development LLC, Spring House, Pennsylvania 19477
Received for publication, December 23, 2002
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ABSTRACT |
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Protease-activated receptor (PAR)-4 is a low
affinity thrombin receptor with slow activation and desensitization
kinetics relative to PAR-1. This study provides novel evidence that
cardiomyocytes express functional PAR-4 whose signaling phenotype is
distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogen-activated protein kinase but is a weak activator of phospholipase C,
extracellular signal-regulated kinase, and cardiomyocyte hypertrophy;
AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1 Serine proteases regulate cell functions in large part by
activating a family of seven transmembrane spanning domain G
protein-coupled receptors
(GPCRs).1 PAR-1, the
prototypical receptor for thrombin, is a ubiquitously expressed GPCR
that is activated by cleavage of its extracellular N terminus to expose
a new N-terminal sequence that binds intramolecularly and serves as a
tethered ligand (1, 2). Since the initial cloning of PAR-1, three
additional structurally homologous PARs have been identified; two newer
PAR family members (PAR-3 and PAR-4) are activated by thrombin, whereas
PAR-2 is activated as a result of limited proteolysis by trypsin,
membrane-type serine protease-1 (a type II transmembrane protein with
serine protease activity) or mast cell tryptase (not by thrombin (3,
4)). Synthetic peptides that mimic the tethered ligand domains of PAR-1 (SFLLRN), PAR-2 (SLIGRL), and PAR-4 (GYPGKF), but not PAR-3, activate their cognate receptors independent of proteolysis (2). Detailed studies identify PAR-1 coupling to several heterotrimeric G protein family members (Gq, Gi, and G12/13)
and thereby a host of intracellular response mechanisms that influence
cell shape, growth, and differentiation. In contrast, knowledge of
PAR-4 expression and function is still quite limited. In heterologous
expression systems, PAR-4 mimics the actions of PAR-1 to activate
phospholipase C and mobilize intracellular calcium. However, major
differences between PAR-1 and PAR-4 in their sensitivity to activation
by thrombin and kinetics of activation and desensitization have been
identified. Specifically, PAR-1 is efficiently cleaved by low
concentrations of thrombin, but PAR-4 activation requires much higher
thrombin concentrations. PAR-4 activation and desensitization kinetics
also are quite slow and sustained relative to the rapid and transient
responses typically elicited by PAR-1 (5). Although these results
suggest that PAR-1 and PAR-4 might not be functionally redundant, there
is still scant information on distinct functional properties of PAR-1 and PAR-4 in native tissues.
The signaling properties of PAR-1 have been explored largely in
platelets (where the actions of thrombin are critical for normal
hemostasis and arterial thrombosis) and the vessel wall (where PAR-1
promotes changes in endothelial cell morphology leading to altered
monolayer permeability and PAR-1 induces proliferation of vascular
smooth muscle cells (2)). Recent studies indicate that cardiomyocytes
represent an additional cardiovascular target for the actions of
thrombin and related proteases. Cardiomyocytes cultured from neonatal
rat ventricles co-express PAR-1 and PAR-2 (mRNA for PAR-3 is not
detected), which stimulate phosphoinositide hydrolysis, activate the
extracellular signal-regulated kinase (ERK) subfamily of
mitogen-activated protein kinases (MAPKs), increase intracellular
calcium, and promote cardiomyocyte hypertrophy (6-10). This study
tests the hypothesis that cardiomyocytes also express PAR-4 and that
PAR-4 activates mechanisms that contribute to cardiac remodeling in
areas of cardiac injury and/or inflammation.
Preparation and Culture of Ventricular Myocytes--
Cardiac
myocytes were dissociated from the ventricles of outbred, PAR-1
knockout, or background strain C57BL/6 mice (embryonic day 18 (11)) by
a trypsin digestion protocol that incorporates a differential
attachment procedure to enrich for cardiac myocytes (6). Although the
preplating step effectively decreases fibroblast contamination,
myocytes were subjected to 30 grays of X-rays on day 1 of culture to
halt the proliferative potential of any residual contaminating
fibroblasts (6). The myocytes were plated at a density of 0.5 × 106 cells/ml (2 ml/35-mm dish) and were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. For assays of ERK, p38-MAPK, or Src activation, the cells were
serum-starved in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12
medium for 24 h.
Reverse Transcriptase-Polymerase Chain Reaction--
Total RNA
was extracted using Trizol Reagent and was reverse transcribed to
random primed cDNA with Superscript reverse transcriptase (Invitrogen). The PCR primers used for PAR-4 were P4Pan-U
5'-GCCAATGGGCTGGCGCTGTG-3' and P4Pan-L 5'- GCCAGGCAGATGAAGGCCGG-3'. The
reactions were carried out in 50-µl reactions using Advantage Klentaq
polymerase (Clontech) with the indicated number of
cycles consisting of a 30-s 94 °C denaturation followed by a 30-s
63.1 °C annealing with a 60-s elongation at 68 °C. The PCR
primers used for Phosphoinositide Hydrolysis--
The cardiomyocytes were
incubated for 72 h with 3 µCi/ml [3H]myoinositol,
washed, preincubated with 10 mM LiCl for 20 min, and then
stimulated with agonists for the indicated intervals at room
temperature. Inositol phosphates (IPs) were extracted and eluted
sequentially by ion exchange chromatography on Dowex columns according
to methods published previously (6).
Src Kinase Activity Assays and Immunoprecipitation for EGFR and
ErbB2--
Src kinase activity was assayed on lysates from cells
extracted for 10 min on ice in extraction buffer (50 mM
Hepes, pH 7.4, 1 mM EGTA, 150 mM NaCl, 1%
Triton X-100, 1% sodium deoxycholate, 10 mM sodium
orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). c-Src was
immunoprecipitated from 550 µg of lysates (precleared with protein
G-Sepharose). Immunoprecipitates were successively washed with
extraction buffer, buffer A (20 mM Tris, pH 7.4, 0.5 mM LiCl, 1 mM EDTA), and buffer B (20 mM Tris, pH 7.4, 10 mM MnCl, 1 mM
EGTA) followed by incubation with 30 µM kinase buffer
containing 20 mM Tris, pH 7.4, 10 mM MnCl, 1 mM EGTA, 5 mM MgCl2, 5 µM cold ATP, and 10 µCi of [32P]ATP with
1 mg/ml enolase as substrate for 15 min at 20 °C. The reactions were
stopped with sample buffer, and the proteins were separated by SDS-PAGE
(10% gel); quantification was with a PhosphorImager (Molecular
Dynamics). The precleared lysates (700 µg) were also subjected to
immunoprecipitation using anti-EGFR and anti-ErbB2 antibodies (Santa
Cruz Biotechnology) followed by immunoblotting with
anti-phosphotyrosine, anti-EGFR and anti-ErbB2 according to the
manufacturer's instructions.
Immunoblot Analysis for ERK and p38-MAPK
Activation--
Activation of ERK and p38-MAPK was monitored by
immunoblot analysis with antibodies for phosphorylated (activated)
signaling proteins. Where indicated, PP1 or AG1478 (Calbiochem) were
included (starting 45 min prior to the stimulatory intervals) to
inhibit the kinase activities of Src family kinases or EGFR kinases,
respectively. In all of the experiments, the immunoblots were stripped
and reprobed with antibodies to total protein to confirm equal protein
loading. Phospho-ERK1/2, total p38-MAPK, and phospho-p38-MAPK
antibodies were from Cell Signaling Technology; antibodies to total
ERK1/2 were from Santa Cruz Biotechnology. Immunoblotting was according to methods published previously or to the manufacturer's instructions (12). Each panel in each figure represents results from a single gel
exposed for a uniform duration, with bands detected by enhanced chemiluminescence and quantified by laser scanning densitometry.
Measurements of Cardiomyocyte Growth--
The measurements of
protein synthesis were performed in triplicate according to methods
published previously (12). Briefly, the cultures were stimulated in
serum-free medium with agonists (or vehicle) for 48 h at 37 °C.
The medium was supplemented with [14C]phenylalanine (0.1 µCi/ml) plus 0.3 mM nonradioactive phenylalanine during
the final 24 h of stimulation. The cells were rinsed with phosphate-buffered saline and incubated in ice-cold 10%
trichloroacetic acid for 30 min. The precipitates were washed twice
with ice-cold 10% trichloroacetic acid and solubilized in 1% SDS at
37 °C for 1 h. Duplicate aliquots from each sample were assayed
for radioactivity and protein content. The results are normalized
to protein content.
Reverse Transcriptase-PCR Analysis of PAR-4--
Total RNA from
cultured embryonic mouse ventricular myocytes (embryonic day
18), cultured neonatal rat ventricular myocytes (postnatal day 2), and
ventricular tissue from adult mouse and rat hearts was converted to
first strand cDNA with reverse transcriptase and used as templates
in analytical PCR reactions. Total RNA from rat platelets was included
as a positive control. The reaction products were analyzed by Southern
blot hybridized with appropriate nested primer probes. Fig.
1 (upper panel) shows that a
band corresponding in size to that predicted by the sequence of the
primer pairs for PAR-4 is detected in all of the samples. The reaction
product is prominent in platelets, but it also is readily detected in samples from adult rodent ventricles and (at lower levels) in neonatal
rat and mouse cardiomyocyte cultures. In cardiomyocyte preparations, a
band that migrates more rapidly (and likely corresponds to a smaller
nested product from internal priming) also is consistently detected.
Equivalent levels of reaction product for PAR-4 Signaling Pathways in Cardiomyocytes--
Consistent with
previous studies establishing functional PAR-1 expression in mouse
cardiomyocyte cultures (10), Fig. 2 shows that SFLLRN induces a rapid/brisk increase in inositol polyphosphates (IP2/IP3) that is followed by a more
progressive and sustained accumulation of IP1. Although
SFLLRN has known agonist activity at both PAR-1 and PAR-2 (13), the
PAR-2-selective agonist peptide SLIGRL does not promote IP accumulation
in mouse cardiomyocytes cultures (data not shown), excluding a
significant contribution of PAR-2 to the actions of SFLLRN. To
determine whether mouse cardiomyocytes express functional PAR-4, the
cultures were stimulated with AYPGKF, a PAR-4-specific agonist peptide
that is reported to be ~10-fold more potent than the PAR-4 tethered
ligand sequence GYPGKF at activating PAR-4 (14). Fig. 2 shows that
AYPGKF promotes phosphoinositide hydrolysis in mouse cardiomyocytes.
However, the characteristics of signaling by AYPGKF-activated PAR-4 and SFLLRN-activated PAR-1 differ in two major respects: 1) the magnitude of IP accumulation (both IP2/IP3 and
IP1) in response to AYPGKF is relatively modest, compared
with the robust responses elicited by SFLLRN and 2) the kinetics of
IP2/IP3 accumulation in response to AYPGKF is
atypically delayed. SFLLRN-induced IP2/IP3
accumulation is detected maximally at early time points (2-5 min);
this response wanes at 30 min. In contrast, little
AYPGKF-dependent IP2/IP3 accumulation is detected at 5 min; a modest
AYPGKF-dependent increase in
IP2/IP3 is detected with longer incubations.
AYPGKF responses cannot be attributed to robust PAR-4 signaling in a
minor contaminating fibroblast population; thrombin and SFLLRN promote
brisk and pronounced increases in IP accumulation in cardiac
fibroblasts, but cardiac fibroblasts do not detectably respond to
AYPGKF (15). Previous studies established that IP accumulation in
response to SFLLRN displays substantial sensitivity to inhibition by
pertussis toxin (PTX; 100 ng/ml for 24 h, a protocol that
completely ADP-ribosylates/inactivates Gi proteins (16)).
In contrast, the modest AYPGKF-dependent activation of
phospholipase C is PTX-insensitive (data not shown).
Fig. 3 compares AYPGKF and SFLLRN
activation of MAPK cascades in cardiomyocytes. AYPGKF activates ERK,
but the magnitude of this response is modest and the kinetics are
protracted relative to the brisk ERK activation induced by thrombin and
SFLLRN. The weak and sluggish nature of PAR-4 signaling to ERK
parallels the protracted time course for PAR-4 agonist activation of
phospholipase C. AYPGKF also activates p38-MAPK. Here, AYPGKF
activation is substantial; it is to a level comparable with p38-MAPK
activation by PAR-1 agonists (or stimuli such as sorbitol, data not
shown). However, the kinetics of p38-MAPK activation by SFLLRN and
AYPGKF differ. p38-MAPK activation by SFLLRN (or thrombin) peaks at 5 min and is sustained for at least 30 min. In contrast, the onset of
p38-MAPK activation by AYPGKF is delayed, with little increase in
p38-MAPK activity in response to AYPGKF during incubations shorter than
20 min. Previous studies identified the effects of thrombin and SFLLRN
to promote cardiomyocyte hypertrophy, as manifest by an
significant increase in [3H]phenylalanine incorporation
into protein as well as total protein content (10). AYPGKF also is a
weak hypertrophic agonist, inducing a 19.3 ± 2.3% increase in
[3H]phenylalanine incorporation into protein (compared
with a 43.7 ± 3.2% increase by thrombin, n = 3 for each, p < 0.05 versus basal).
Src has been implicated in many of the cellular actions of thrombin. To
determine whether Src contributes to cardiomyocyte activation by
thrombin, the cardiomyocytes were exposed to vehicle or PAR agonists,
and the intrinsic kinase activity of Src was determined by an immune
complex kinase assay. Fig. 4A
shows that thrombin increases the kinase activity of Src toward the
exogenous substrate enolase. In contrast, Src is not activated by
SFLLRN. This result is surprising, because thrombin-dependent
activation of Src generally is ascribed to a signaling pathway
emanating from PAR-1 (which would be activated by SFLLRN). This
pharmacologic profile suggested that Src might lie downstream from
PAR-4 (not PAR-1) in cardiomyocytes. Fig. 4A shows that
AYPGKF induces a modest level of Src activation, consistent with this
formulation.
The PAR-1 requirement for thrombin-dependent activation of
Src was explored further in cardiomyocytes cultured from
PAR-1
To determine whether Src lies upstream from p38-MAPK in the PAR-4
signaling pathway, further studies were performed with PP1 (a specific
inhibitor of Src family tyrosine kinases). Fig.
6 shows that PP1 blocks p38-MAPK and ERK
activation by AYPGKF; this is not due to a nonspecific inhibitory
effect of PP1, because ERK and p38-MAPK activation by sorbitol is
equivalent in control and PP1-treated cultures. Given the recent
evidence that hypertrophic signaling by cardiomyocyte G protein-coupled
receptors also can involve EGFR transactivation (17), AYPGKF responses
also were examined in cultures pretreated with AG1478 (a well
established inhibitor of the kinase activity of the EGFR (18, 19)).
Fig. 6 shows that AG1478 also inhibits ERK and p38-MAPK activation by
AYPGKF (but not sorbitol) in cardiomyocytes. Separate experiments demonstrated that PAR-4 activation of p38-MAPK and Src is not blocked
by PTX (data not shown). Collectively, these results implicate Src and
EGFR kinases activities, but not Gi proteins, in the
pathway for PAR-4-dependent activation of ERK and p38-MAPK
in cardiomyocytes.
PAR-4 Signaling in Cells That Stably Overexpress PAR-4--
PAR-4
expression reconstitutes thrombin-dependent activation of
ERK and p38-MAPK in fibroblasts from PAR-1
Fig. 7 shows that thrombin induces a robust increase in ERK and
p38-MAPK activity at 5 min; ERK activation wanes slightly at 30 min,
whereas p38-MAPK activation persists during this interval. PP1 is a
generalized inhibitor of thrombin-dependent activation of
ERK and p38-MAPK at both early and late time points. This result implicates Src kinases in all aspects of PAR-4 signaling. At the early
time point, thrombin-dependent activation of ERK also is blocked by GF109203X; GF109203X does not inhibit ERK activation at the
later time point or thrombin-dependent activation of
p38-MAPK at either time point. The PAR-4 signaling phenotype in
GF109203X-treated cells is reminiscent of PAR-4 signaling in
cardiomyocytes, where AYPGKF induces prominent p38-MAPK activation and
ERK activation with delayed kinetics.
The more delayed component of thrombin-dependent activation
of ERK and p38-MAPK is blocked by the EGFR antagonist AG1478 or PP1
(and not by GF109203X). Thrombin-dependent activation of
ERK at 30 min is inhibited by PP1 and AG1478, whereas
EGF-dependent activation of ERK is blocked only by AG1478
(not by PP1). This result establishes the efficacy of inhibition by
AG1478, as well as the specificity of PP1 for Src (and not the EGFR).
This result also places Src upstream from EGFRs in the PAR-4 signaling
pathway leading to ERK activation (although further evidence that PP1 effectively blocks EGF-dependent activation of p38-MAPK
indicates that Src also exerts a role downstream from EGFRs in the
pathway leading to p38-MAPK activation).
The pharmacologic studies implicate EGFR transactivation in the PAR-4
signaling pathway. These results are quite novel; there is precedent
for PAR-1 transactivation of EGFR and the related EGFR family member
ErbB2 (20), but EGFR transactivation as a mechanism for signaling by
PAR-4 has never been reported. Because preliminary studies indicated
that PAR-4-expressing fibroblasts from PAR-1
Collectively, these studies effectively resolve two parallel
Src-dependent signaling pathways emanating from PAR-4 (as
schematized in Fig. 9). The initial rapid
PAR-4-dependent activation of ERK involves
Gq/phospholipase C-dependent activation of
protein kinase C and Src. The more sustained
PAR-4-dependent activation of ERK (and p38-MAPK) is via a
separate Src-dependent pathway that involves EGFR and/or
ErbB2 transactivation. This latter Src-p38-MAPK pathway dominates in
cardiomyocytes.
These studies provide novel evidence that cardiomyocytes express
functional PAR-4 and that the signaling properties of PAR-4 in
cardiomyocytes are distinct from those previously reported for PAR-1.
Specifically, PAR-1 promotes the rapid/strong activation of
phospholipase C and ERK (via PTX-sensitive and -insensitive pathways)
and a more sustained activation of p38-MAPK. Although human PAR-4
(expressed in PAR-1 This study provides surprising evidence that
thrombin-dependent activation of Src is mediated by PAR-4
(and not PAR-1) in cardiomyocytes. It is noteworthy that many
laboratories have reported effects of thrombin to activate Src family
kinases. Although an early study performed on the CCL39 Chinese hamster
fibroblast line implicated PAR-1 in thrombin-dependent
activation of Src (on the basis of evidence that a PAR-1 agonist
peptide mimics the effect of thrombin to activate Src (22)), most
subsequent studies have focused on Src family kinase activation by
thrombin in platelets and have not attempted to replicate the response with a PAR-1 agonist peptide. In this context, the studies reported herein demonstrate that thrombin activates Src in cardiomyocytes and
that this response is mimicked by the PAR-4 (not the PAR-1) agonist
peptide. The observation that thrombin activates Src in PAR-1 This study identifies a Src-p38-MAPK pathway emanating from PAR-4 in
mouse cardiomyocytes; the PAR-4 agonist peptide is a rather weak
hypertrophic agonist relative to other hypertrophic stimuli such as
thrombin, norepinephrine, or endothelin. Of note, Src and p38-MAPK on
balance are implicated in deleterious functional and structural changes
in cardiomyocytes (25). The Src-p38-MAPK pathway is opposed by ERK
(which is generally considered cardioprotective) when the actions of
thrombin are mediated by the combined actions of PAR-4 and PAR-1.
However, the conditions that render PAR-1 inactive (such as
inflammation, where neutrophil-derived proteases such as cathepsin G
would amputate the N-terminal tethered ligand domain of PAR-1) would
shift the balance of signaling by PAR-1 and PAR-4, leading to unopposed
PAR-4 activation. Under these conditions, dominant signaling through
Src and p38-MAPK may contribute to adverse functional outcomes.
The predominant natural activator(s) of endogenous cardiomyocyte PARs
remains uncertain. Although cardiomyocyte PAR-1 may be activated by
thrombin in the setting of hemorrhagic infarction (where the
endothelial barrier is broken and cardiomyocytes come into direct
contact with blood-borne substances), most myocardial events are not
accompanied by hemorrhage into the myocardium. Hence, other potential
mechanisms for PAR activation must be considered. PAR-4 has been
identified as a potential substrate for neutrophil-derived proteases
that are released at sites of cardiac injury and/or inflammation (26).
Hence, the cardiac actions of PAR-4 may become important in the context
of cardiac remodeling during myocarditis and/or at the border zone of a
myocardial infarction.
/
mice establishes that Src
activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further
studies implicate Src and epidermal growth factor receptor (EGFR)
kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1
/
cells that stably overexpress PAR-4; the
Src-mediated pathway for EGFR/ErbB2 transactivation underlies the
protracted phases of thrombin-dependent extracellular
signal-regulated kinase activation in PAR-1
/
cells that
overexpress PAR-4 and in cardiomyocytes. These studies identify a
unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G
protein-coupled receptors) that is predicted to contribute to cardiac
remodeling and influence the functional outcome at sites of cardiac inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin were
-actin PTP-U
5'-AGGCCAACCGCGAGAAGATG-3' and
-actin PTP-L
5'-CTCGGCCGTGGTGGTGAAGC-3'. The
-actin reactions were carried out in
25-µl reactions using Advantage Klentaq polymerase
(Clontech) for 25 cycles consisting of a 30-s
94 °C denaturation followed by a 30-s 60.4 °C annealing with a
30-s elongation at 68 °C. The amplified products were fractionated in 2% agarose gels and Southern transferred to Hybond N+ membrane (Amersham Biosciences). The blots were hybridized to the appropriate digoxigenin (DIG)-labeled nested primer probes; PANP4 PP-L
5'-CCAGCAGCAACACTGAACCATACATGTGGCCATAGAG-3' for PAR-4, and actin
PP-L 5'-TGGGCACAGTGTGGGTGACCCCGTCACCGGAGTCCATCAC-3' for
-actin. The specific PCR products were then visualized using DIG luminescent detection (Roche Molecular Biochemicals).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin verify uniform
loading, and
-actin is detected only in reactions that include
reverse transcriptase performed in parallel as negative controls. These
studies identify PAR-4 transcripts in mouse and rat cardiomyocytes.
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Fig. 1.
Southern blot analysis of PCR reactions for
PAR-4. PCR reactions were carried out in the presence (+) or
absence ( ) of reverse transcriptase (RT) on cardiomyocytes
cultured from day 18 embryonic mouse ventricles, postnatal day 2 rat
ventricles, adult mouse and rat ventricular myocardium, and rat
platelets. Amplification was for 25 cycles for
-actin or as
indicated for PAR-4, with specific amplification products denoted by
the arrows. Trace PAR-4 product is detected in reactions
without reverse transcriptase, suggesting trace background
contamination of samples with genomic DNA. Normalization is to
-actin, which is detected at uniform levels and only when reverse
transcriptase was present in the reaction.
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Fig. 2.
SFLLRN and AYPGKF activation of PLC in
cardiomyocytes. Stimulation was with SFLLRN (300 µM)
or AYPGKF (500 µM) for 5 or 30 min, with IP metabolites
sequentially eluted by Dowex column chromatography according to
standard methods. The results are expressed as cpm over basal
(mean ± S.E.) for triplicate determinations from four separate
culture preparations. The effects of SFLLRN on IP accumulation at both
time points are significant; AYPGKF significantly increases
IP1 at both time points and IP2 + IP3 at 30 min (p < 0.05 compared with
basal).
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Fig. 3.
AYPGKF and SFLLRN activate ERK1/2 and
p38-MAPK cascades in cardiomyocytes. Serum-starved cultured
cardiomyocytes were treated with vehicle, AYPGKF (500 µM), or SFLLRN (300 µM) for the indicated
intervals or thrombin (at 1 or 10 units/ml for 5 min in the top
panel and 1 unit/ml for 5 and 30 min in the bottom
panel). The cell lysates were subjected to SDS-PAGE and Western
blotting with anti-phospho-ERK1/2 or anti-phospho-p38-MAPK antibodies.
Immunoblot analysis with total ERK1/2 and p38-MAPK established constant
protein loading in all lanes (data not shown). Representative
autoradiograms (with each lane from a single gel exposed for the same
duration) are presented. Similar results were obtained in three
separate experiments. CT, control; P,
phospho.
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Fig. 4.
Src activation by thrombin and AYPGKF in
cardiomyocytes from wild type and PAR-1 /
mice. Serum-starved cardiomyocytes were exposed to vehicle,
thrombin (1 unit/ml), SFLLRN (300 µM), or AYPGKF (500 µM) for 10 min. Src activity with enolase as substrate
was measured as described under "Materials and Methods."
Representative experiments are illustrated in A and
B, with results obtained in three separate culture
preparations quantified in C. WT, wild
type.
/
mice. This model was first validated by
demonstrating that SFLLRN and the purinergic agonist ATP activate
phospholipase C and ERK in wild type cardiomyocytes, but only ATP
activates phospholipase C and ERK in PAR-1
/
cardiomyocytes (Fig. 5, A and
B). Although PAR-1
/
cardiomyocytes are
unresponsive to SFLLRN, effects of thrombin to activate p38-MAPK and
Src persist in PAR-1
/
cardiomyocytes (Figs. 4,
B and C, and 5C);
AYPGKF-dependent activation of p38-MAPK and Src also is
detected in this preparation. Collectively, these results indicate that
cardiomyocyte responses to thrombin are mediated by the combined
actions of PAR-1 and PAR-4. PAR-1 activates the phospholipase C/ERK
pathway and also provides a mechanism to activate p38-MAPK. In
contrast, PAR-4 promotes only a low level of sustained phospholipase
C/ERK activation; its more prominent action is to stimulate p38-MAPK
and Src. Surprisingly, thrombin-dependent activation of Src is
mediated only by PAR-4 in cardiomyocytes (and not PAR-1).
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Fig. 5.
SFLLRN activation of phospholipase C and ERK
is lost, but thrombin and AYPGKF activation of p38-MAPK persists, in
PAR-1 /
cardiomyocytes. Cardiomyocytes cultured
from wild type and PAR-1
/
ventricles were stimulated
with vehicle, SFLLRN (300 µM), or ATP (100 nM) for 30 min (A) or 5 min (B).
A, inositol phosphate accumulation was determined by Dowex
column chromatography, and ERK activation was identify by SDS-PAGE and
Western blotting of cell lysates with anti-phospho-ERK1/2 antibody. The
control experiments established that ERK protein expression is
equivalent in wild type and PAR-1
/
cultures.
B, incubations were with AYPGKF (500 µM) or
thrombin (at 1 or 10 units/ml) for the indicated intervals, with
p38-MAPK activation tracked with an antibody specific for the
phosphorylated (activated) species. The results are the means ± S.E. from triplicate determination in three separate cultures
(A) or representative autoradiograms (with each lane from a
single gel exposed for the same duration) from a single experiment,
with equivalent results in two other culture preparations, in
B and C. WT, wild type; P,
phospho.
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Fig. 6.
PAR-4-dependent stimulation of
ERK and p38-MAPK requires intact Src and EGFR kinase activity in
cardiomyocytes. Serum-starved cardiomyocyte cultures from outbred
mice were treated with vehicle, AYPGKF (500 µM), or
sorbitol (0.5 M) for 30 min without or with PP1 (10 µM) or AG1478 (2 µM; Calbiochem)
pretreatment, starting 45 min prior to stimulation. The efficacy of the
AG1478 pretreatment protocol was established in separate experiments
demonstrating complete inhibition of ERK and AKT activation by
heregulin (data not shown). Western blotting was with the
anti-phospho-p38-MAPK or anti-phospho-ERK antibodies; immunoblot
analysis with total ERK1/2 and p38-MAPK established constant protein
loading in all lanes (data not shown). Similar results were obtained in
three separate experiments. P, phospho.
/
mice (Fig.
7), providing a model system to resolve
the mechanism(s) underlying the actions of PAR-4. The goal of studies
in PAR-4-expressing cells was to determine whether separate signaling
pathways for PAR-4 could be resolved (and whether PAR-4 signaling in
cardiomyocytes conforms to a defined subset of the actions of PAR-4).
GF109203X was used to inhibit phorbol ester-sensitive protein kinase C
isoforms and thereby ablate the phospholipase C-protein kinase C
pathway. PP1 and AG1478 were used to inhibit the kinase activities of
Src and EGFRs, respectively. The EGFR antagonist was included in these experiments as a control for the specificity of inhibition by PP1; it
also provided a strategy to consider a potential role for EGFR
transactivation in PAR-4 signaling.
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Fig. 7.
The roles of protein kinase C, Src, and EGFR
transactivation in PAR-4-dependent stimulation of ERK and
p38-MAPK in clonal derivatives of PAR-1 /
fibroblasts
that stably overexpress PAR-4. The cells were serum-starved and
then challenged with vehicle, thrombin (10units/ml for 5 or 30 min), or
EGF (100 ng/ml for 5 min) without or with pretreatment with GF109203X
(5 µM), PP1 (10 µM), or AG1478 (2 µM) each starting 45 min prior to stimulation. Western
blotting was with the anti-phospho-ERK1/2 or anti-phospho-p38-MAPK
antibodies. A representative experiment is depicted, with each lane
from a single gel exposed for a uniform duration. Equivalent results
were obtained in three separate experiments. P,
phospho.
/
mice
express EGFRs and ErbB2 (data not shown), we explored the potential
individual roles for EGFRs and ErbB2 in the PAR-4-dependent signaling pathway. Fig. 8 shows that
thrombin promotes a large increase in the tyrosine phosphorylation of
~170-175- and ~180-185-kDa proteins, respectively, in anti-EGFR
and anti-ErbB2 immunoprecipitates from PAR-4 expressing
PAR-1
/
fibroblasts; thrombin-dependent
tyrosine phosphorylation is largely blocked by the Src kinase inhibitor
PP1 (but not by PP3, a structurally analogous compound that does not
inhibit Src family kinases, attesting to the specificity of the
inhibitory actions of PP1; data not shown). The direct
activation/phosphorylation of EGFR (by EGF) and ErbB2 (by EGF and
heregulin) is not blocked by PP1; to the contrary,
EGFR-dependent tyrosine phosphorylation of ErbB2 is slightly increased, and heregulin-induced tyrosine phosphorylation of
ErbB2 is markedly increased in PP1-treated cultures. A similar effect
of PP1 to increase EGF phosphorylation of ErbB2 was identified previously (21). The blots were stripped and reprobed with anti-EGFR and ErbB2 to validate equal protein loading. EGFR immunoreactivity is
detected at grossly similar levels in all of the samples. ErbB2 also is
detected at similar levels in samples from unstimulated, thrombin-stimulated, and heregulin-stimulated cultures, but ErbB2 protein appears to be reduced in EGF-stimulated samples (and in the
EGF- and heregulin-stimulated samples from PP1-treated cultures). However, this is an artifact, related to the sequence for
immunoblotting in this experiment, because ErbB2 is detected at similar
levels in all samples when the immunoblot is first probed with
anti-ErbB2 antibody (data not shown). This suggests that extensive
signals with the anti-phosphotyrosine antibody prevent subsequent
anti-ErbB2 antibody binding to the ErbB2 protein epitope.
View larger version (83K):
[in a new window]
Fig. 8.
Thrombin promotes EGFR and ErbB2
phosphorylation in clonal derivatives of PAR-1 /
fibroblasts that stably overexpress PAR-4. The cells were
serum-starved and then challenged with vehicle, thrombin (10 units/ml
for 10 min), EGF (100 ng/ml for 5 min), or heregulin (10 nM
for 5 min) without or with pretreatment with PP1 (10 µM).
The extracts were subjected to immunoprecipitation (IP) with
anti-EGFR (top panels) or anti-ErbB2 (bottom
panels), followed by immunoblot (IB) analysis with
anti-phosphotyrosine; the blots were stripped and then reprobed for
EGFRs (top panels) or ErbB2 (bottom panels). A
representative experiment is depicted, with each lane from a single gel
exposed for a uniform duration. Equivalent results were obtained in
three separate experiments.
View larger version (37K):
[in a new window]
Fig. 9.
Schematic of PAR-4 signaling
pathways.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
fibroblasts) supports
thrombin-dependent activation of all of these responses
(phospholipase C, ERK, and p38-MAPK), PAR-4 native to mouse
cardiomyocytes preferentially elicits a subset of these responses.
PAR-4 stimulation leads to a relatively strong activation of p38-MAPK,
with only a minor associated increase in phospholipase C and ERK that
is delayed in onset. Additional studies implicate tyrosine
phosphorylation events on both EGFRs and ErbB2 in the actions of PAR-4,
providing the first evidence that transactivation of EGFR family
members can contribute to PAR-4 responses. The protracted kinetics for
PAR-4 signaling in cardiomyocytes presumably is attributable to the
slow activation and desensitization kinetics described for PAR-4
(relative to PAR-1 (5)). However, a mechanism that accounts for the
very weak PAR-4 coupling to the phospholipase C/ERK pathway is not
obvious. It is tempting to speculate that this lesion in PAR-4
signaling in mouse cardiomyocytes is part of a more generalized
defect in GqPCR-dependent activation of phospholipase C recently identified in mouse cardiomyocytes;
1-adrenergic agonists and endothelin promote only a
trivial activation of phospholipase C in mouse cardiomyocytes compared
with the robust actions of these agonists in rat cardiomyocytes (16).
According to this formulation, PAR-1 activation of phospholipase C is
preserved in mouse cardiomyocytes, because it is largely mediated by
PTX-sensitive G proteins and not Gq; PAR-4 coupling through
Gq to phospholipase C is impaired. Alternatively,
differences in PAR-4 signaling may not necessarily reflect a limitation
of the signaling machinery inherent to mouse cardiomyocytes but could
be due to species-dependent differences in the signaling
properties of the expressed human PAR-4 and the native PAR-4 sequence
in mouse cardiomyocytes.
/
cardiomyocytes provides further evidence that
Src activation does not require PAR-1 expression. The mechanism whereby
a GPCR such as PAR-4 activates Src family tyrosine kinases has been the focus of research by several laboratories. Although Src activation by
GPCRs has been attributed in certain model systems to the downstream effectors of heterotrimeric Gq proteins (calcium,
direct actions of PKC, or indirect actions of PKC to stimulate PYK2
(23)), the relatively weak activation of phospholipase C by PAR-4 makes this pathway unlikely. Similarly, effectors downstream from G protein
dimers (such as phosphatidylinositol 3'-kinase) have been
implicated in Src activation (24). Because
dimers generally are
derived from Gi proteins, this pathway also is unlikely
because PAR-4 signaling is PTX-insensitive. Other mechanisms for Src
activation (including a direct interaction of Src with G protein
subunits, anchoring of Src to a signaling complex formed by the GPCR
and the scaffolding protein
-arrestin, and/or anchoring of Src
directly to the agonist-activated GPCR) should be considered in future studies.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Ema Stasko for preparing myocyte cultures.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service-NHLBI, National Institutes of Health Grant HL-64639.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.
§ Present address: University of Alabama, Birmingham
To whom correspondence should be addressed: Dept. of
Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168 St., New York, NY 10032. Tel.: 212-305-4297; Fax: 212-305-8780; E-mail: sfs1@columbia.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M213091200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G protein-coupled receptor; PAR, protease-activated receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; IP, inositol phosphate; EGFR, epidermal growth factor receptor; IP2, inositol 1,4-bisphosphate; IP3, inositol 1,4,5-trisphosphate; PTX, pertussis toxin.
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