From the Department of Pharmacology, College of
Medicine, University of Illinois, Chicago, Illinois 60612 and the
¶ Department of Molecular Pharmacology and Biological Chemistry,
Northwestern University School of Medicine,
Chicago, Illinois 60611
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ABSTRACT |
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We addressed the mechanisms of restoration of
cell surface proteinase-activated receptor-1 (PAR-1) by investigating
thrombin-activated signaling pathways involved in PAR-1 re-expression
in endothelial cells. Exposure of endothelial cells transfected with
PAR-1 promoter-luciferase reporter construct to either thrombin or
PAR-1 activating peptide increased the steady-state PAR-1 mRNA and
reporter activity, respectively. Pretreatment of reporter-transfected
endothelial cells with pertussis toxin or co-expression of a minigene
encoding 11-amino acid sequence of COOH-terminal
G Endothelial cell responsiveness to thrombin requires the
expression of cell surface proteinase-activated receptor-1
(PAR-1)1 (1-6). Activation
of PAR-1 by cleavage of the NH2 terminus of PAR-1 results
in changes in endothelial cell function such as expression of
endothelial cell adhesion molecule ICAM-1 and increased endothelial
permeability (7-9). The regulatory mechanisms underlying PAR-1 gene
expression are not well characterized. Thrombin-induced PAR-1 cleavage
leads to PAR-1 endocytosis and degradation in lysosomes (10);
therefore, the activated PAR-1 is unable to recycle to the cell
membrane and resensitize cells to thrombin as with other G
protein-coupled receptors (10). Previous studies have shown that
endothelial cells also contain a preformed pool of PAR-1 that
translocates within minutes to the cell surface in response to thrombin
(10, 11). This protein synthesis-independent PAR-1 expression confers
thrombin sensitivity by rapidly replenishing the cell surface PAR-1
(10); however, this pool has a limited and short-lived ability to
resensitize endothelial cells to thrombin (5). Further, thrombin
responsiveness required de novo PAR-1 synthesis (5),
indicating that it is necessary to activate synthesis of PAR-1 to
regenerate fully the cell surface PAR-1 population once the preformed
receptor pool has been depleted.
PAR-1 has been shown to couple functionally with multiple
heterotrimeric G proteins, which may in part explain the pleiotropic actions of thrombin (12, 13). In endothelial cells, PAR-1 binds to
heterotrimeric Gi and Gq, and can thus initiate
downstream signaling events (13). As receptors coupled to
Gi activate the Ras/MAPK cascade, which in turn results in
gene transcription (14-18), in the present study, we investigated the
possible role of Gi activation of Ras/MAPK pathway in
regulating thrombin-induced PAR-1 gene expression in endothelial cells.
Materials--
Dulbecco's modified Eagle's medium (DMEM) and
diethyl pyrocarbonate were purchased from Sigma. Fetal bovine serum
(FBS) was obtained from HyClone, Logan, UT. Duralose membrane,
Quickhyb, and Prime-It II were from Stratagene, La Jolla CA. PAR-1
activating peptide, SFLLRNPNDKYEPF (TRP-14) was synthesized and
purified as described (4). An ~3-kb 5'-regulatory portion of cloned PAR-1 gene (19, 20) subcloned into pBluescript was obtained from Dr.
W. F. Bahou (SUNY, Stony Brook, NY). TRIzol reagent, polymerase
chain reaction (PCR) primers, green fluorescent protein (Green
Lantern-1) plasmid DNA, LipofectAMINE, and Opti-MEM I were from Life
Technologies Inc.. The reporter vectors pGL2 (firefly, Photinus
pyralis) and pRL (sea pansy, Renilla reniformis)
luciferase and the Dual Luciferase Reagent Assay System were from
Promega Corp., Madison, WI. Protein assay reagents were from
Bio-Rad. Genistein,
4-amino-5-(4methyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), wortmannin, LY294002, and anti-v-Src antibody were from Calbiochem-Novabiochem, La Jolla, CA. Pertussis toxin was from List
Biologicals, Campbell, CA. Anti-Erk1, anti-p85 phosphoinositide 3-kinase (PI3K), and anti-Shc antibodies were from Transduction Laboratories, Lexington, KY. The anti-hemagglutinin (HA) antibody was
from Aves Labs, Tigard, OR. Phosphospecific Erk1 was from Promega Corp.
Plasmid DNA encoding mutant forms of MEK (HA-tagged MKK1 in pCEP4),
CAAX-Raf (in pCEXV3), G Endothelial Cell Cultures--
Human microvessel endothelial
cells (a human dermal microvascular endothelial cell line) were
obtained from Dr. Edwin W. Ades (National Center for Infectious
Diseases, Center for Disease Control, Atlanta, GA) (24). The cells were
grown in endothelial basal medium MCDB-131 supplemented with 10% FBS,
2 mM L-glutamine, and 1 mg/ml hydrocortisone.
Human pulmonary artery endothelial cells (HPAEC) obtained from
Clonetics Corp. (San Diego, CA) were grown in EBM-2 medium supplemented
10% FBS. Cells were cultured on tissue culture dishes coated with
0.1% gelatin. Cells were used between passages 4 and 8.
Northern Blot Analysis--
Endothelial cells were incubated in
serum-free DMEM for 2 h prior to agonist exposure for the
indicated times. Total cellular RNA was extracted using TRIzol reagent
per manufacturer's instructions. Briefly, following treatment, cells
were lysed using 2 ml of TRIzol reagent/100-mm2 tissue
culture dish. Chloroform (0.2 volume) was added to cell lysate, and the
aqueous phase was collected by centrifugation. The total cellular RNA
from the aqueous phase was precipitated with an equal volume of
isopropanol for 1-2 h at Construction of PAR-1 Reporter Plasmid--
A 1.82-kb PAR-1
promoter sequence was made by PCR amplification of directly upstream of
the initiation codon from an ~3.0-kb exon I containing PAR-1 gene
5'-untranslated region (19). The proximal primer incorporated a
HindIII restriction site into the sequence for subcloning
purposes (proximal primer 3'-aagcttttgtcccgggctctgcgcggcgctgc-5'; distal primer 5'-aagctttatttaactgggtacttcc-3'). The amplified fragment
(PCR-1) was inserted into pUC118 for amplification and restriction
analysis. This plasmid, PCR-1/pUC118, was digested with
KpnI/BglII to release PCR-1 with cohesive ends
compatible with KpnI/BamHI-digested pGL2 to form
PCR-1/Luc. The sea pansy (R. reniformis) luciferase gene
driven by herpes simplex virus-thymidine kinase promoter (TK/pRL)
served as an internal control to correct for experimental variation.
Transfections--
HMEC, grown in six-well plates to 50-70%
confluence, were incubated with LipofectAMINE-DNA complexes for 2-4 h.
LipofectAMINE-DNA complexes were made by incubating 4 µg of
LipofectAMINE with 0.1-1.0 µg of plasmid DNA in 0.2 ml of Opti-MEM I
for 45 min at 22 °C. LipofectAMINE-DNA complexes were diluted with
0.8 ml of Opti-MEM I before being added to HMEC, prewashed two times
with Opti-MEM I, for 2-4 h. To end transfection, 2 ml of MCDB 131 medium supplemented with 10% FBS was added to each well. We determined
the transfection efficiency in HMEC using green fluorescent protein
(GFP) plasmid, which encodes a mutated form of the GFP gene driven by a
cytomegalovirus immediate early promoter. HMEC transfected with GFP
plasmid were visualized by fluorescence microscopy with a blue filter
(peak excitation 490 nm). Approximately 30% of HMEC appeared green
24 h after being transfected with GFP plasmid DNA.
Dual Luciferase Reporter Assay--
After transfection, the
cells were incubated in growth medium for 20-24 h. Cells were then
incubated in serum-free DMEM for 2 h prior to addition of agonist.
Following stimulation, cells were lysed (according to manufacturer's
instructions) and 20 µl of lysate (500 µl total) was assayed for
reporter gene expression. Firefly (P. pyralis) and sea pansy
(R. reniformis) luciferase activity were assessed by the
Dual Luciferase Reagent Assay System. Protein concentrations were
determined using Bio-Rad reagents.
p44/42 MAPK Assay--
MAPK activity was measured by using the
MAPK assay kit from New England Biolabs, Inc. (Beverly, MA).
Endothelial cell monolayers in 100-mm2 culture dishes were
treated with or without agonist for the indicated times at 37 °C.
Cells were washed three times with phosphate-buffered saline and lysed
with lysis buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1.0% Triton X-100, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate, 1 mM Immunoprecipitation--
Endothelial cell monolayers in
100-mm2 culture dishes were treated with agonist for the
indicated times at 37 °C. Cells were washed three times with
phosphate-buffered saline and lysed in lysis buffer (50 mM
Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM
EDTA, 0.25% sodium deoxycholate, 1.0% Nonidet P-40, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotinin, and 44 µg/ml PMSF) for 30 min at 4 °C. Insoluble material was removed by
centrifugation (13,000 × g for 15 min) prior to
overnight immunoprecipitation with 1 µg/ml antibody (as indicated) at
4 °C. Protein A- or G-agarose beads were added to each sample and
incubated for 1 h at 4 °C. Immunoprecipitates were gently
washed three times with wash buffer (Tris-buffered saline containing
0.05% Triton X-100, 1 mM Na3VO4, 1 mM NaF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotinin, and 44 µg/ml PMSF). Immunoprecipitated proteins
were resolved on SDS-PAGE and transferred to Duralose membrane. The
membrane was utilized for immunoblotting with indicated antibodies
(described below).
Western Blot Analysis--
Endothelial cell lysates or
immunoprecipitates were resolved by SDS-PAGE on a 12% separating gel
under reducing conditions and transferred to Duralose membrane.
Membranes were blocked with (5% dry milk in 10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 2 h at 22 °C. Membranes were incubated with indicated primary antibody
(diluted in blocking buffer) at 22 °C for at least 1 h.
Following washes, membranes were incubated at 22 °C with horseradish
peroxidase-conjugated goat anti-rabbit or mouse antibody. Protein bands
were detected by ECL method.
Endothelial Cell Shape Change Assay--
The time course of
endothelial cell shape change (measured in real time) in response to
thrombin was determined according to procedures described by us (25).
In brief, HPAEC were grown to confluence on small gold electrodes
(4.9 × 10 Statistical Analysis--
Statistical comparisons were made
using two-tailed Student's t test. Experimental values were
reported as mean ± S.E. Differences in mean values were
considered significant at p < 0.05.
Thrombin-induced PAR-1 Activation Increases Steady-state PAR-1
mRNA--
To determine if PAR-1 activation increases the PAR-1
mRNA levels in endothelial cells, Northern blot analysis was
performed on total RNA isolated from agonist-stimulated cells (see
"Experimental Procedures"). Confluent endothelial cell monolayers
were stimulated for the indicated times with PAR-1 agonists, thrombin,
or the 14-amino acid PAR-1 tethered ligand, TRP-14 (12). The expression of GAPDH was unaffected by the experimental conditions and was thus
used an internal control. In primary cells, the HPAEC, PAR-1/GAPDH ratio increased 2-fold (over untreated controls) after thrombin treatment (Fig. 1A). This
increase was sustained through 6 h of thrombin treatment. A
similar response was observed with thrombin treatment of HMEC (Fig.
1B). Stimulation of HPAEC with TRP-14 also increased
PAR-1/GAPDH 1.9-fold by 6 h (Fig. 1C).
Thrombin-mediated Transactivation of PAR-1 Promoter--
To
explore the induction of PAR-1 gene, the PAR-1 promoter reporter
plasmid (PAR-1/Luc) and TK/pRL (see "Experimental Procedures") were
transiently expressed in HMEC. Cells were challenged with either
thrombin or TRP-14 for various times and lysed, and luciferase activity
was measured (see "Experimental Procedures"). Luciferase activity
significantly increased over basal at 2 h after thrombin stimulation. By 4-6 h of thrombin exposure, luciferase levels were
~3-fold greater than in untreated cells. After 10 h of thrombin exposure, luciferase activity showed a downward trend (Fig.
2). Exposure of reporter
plasmid-transfected HMEC with different concentrations of thrombin
ranging from 10 nM to 300 nM did not alter the
expression of reporter (data not shown). Luciferase activity (Fig. 2)
and PAR-1 mRNA levels (Fig. 1) were increased over a similar time course with maximal gene expression occurring between 3 and 6 h.
To determine whether PAR-1 activation itself is sufficient to induce
the PAR-1 gene, HMEC transfected with the reporter plasmid were exposed
to TRP-14. Luciferase activity increased >2.5-fold over basal after
4 h of 100 mM TRP-14 stimulation, indicating that
activation of endothelial cell surface PAR-1 is responsible for the
induction of PAR-1 gene expression.
Pertussis Toxin-sensitive G Proteins Mediate Thrombininduced
PAR-1 Gene Expression--
To determine the role of Gi in
thrombin-mediated PAR-1 gene induction, HMEC expressing the reporter
construct were pretreated with pertussis toxin (PTX) to uncouple PAR-1
from PTX-sensitive G proteins. PTX pretreatment prevented the increase
in luciferase activity by 90%, following thrombin stimulation (Fig.
3). Basal luciferase levels were not
altered by PTX pretreatment. Thus, PTX-sensitive G proteins are
critically involved in mediating thrombin-induced PAR-1 gene
expression.
The interface between the G
Receptors coupled to PTX-sensitive Gi proteins have been
shown to activate downstream signal transduction pathways in both PTX Prevents p42/44 MAPK Activation--
Because MAPK regulates
gene transcription (30), we determined if MAPK contributes to the
downstream signaling required for PAR-1 gene expression. Thrombin
exposure in HMEC caused a rapid and transient phosphorylation of MAPK,
which was maximal at 2 min (Fig.
5A). Pretreatment of HMEC with
PTX prevented the thrombin-induced MAPK phosphorylation at 2 min (Fig.
5B). However, PTX pretreatment did not inhibit the
phosphorylation of MAPK by
12-O-tetradecanoylphorbol-13-acetate. Activation of MAPK was
also measured by its ability to phosphorylate Elk-1 in
vitro (see details under Experimental Procedures") as the
phosphorylation of Ser-383 on Elk-1 by MAPK is required for Elk-1-dependent gene transcription (31). We showed that PTX pretreatment prevented the MAPK-mediated Elk-1 phosphorylation after
thrombin stimulation. In contrast, basic fibroblast growth factor-mediated Elk-1 phosphorylation was PTX-insensitive (Fig. 5C). Thus, activation of Gi/o mediated the
thrombin-induced MAPK phosphorylation and activation of MAPK.
Ras-activated Pathway Mediates Thrombin-induced PAR-1
Expression--
To address the role of Ras and Ras-activated
pathway in thrombin-induced PAR-1 gene expression,
luciferase-reporter constructs were co-expressed with dominant-negative
Ras (N17Ras) in HMEC. In N17Ras-expressing HMEC, PAR-1 driven
luciferase activity was reduced by 51% following thrombin treatment
(Fig. 6A). Moreover, the
oncogenic form of Ras, V12Ras, activated PAR-1 gene transcription independent of stimulation by thrombin (Fig. 6A).
Since an immediate downstream target of Ras is the serine/threonine
kinase Raf, we tested the ability of CAAX-Raf, which
constitutively localizes to the cytosolic face of plasma membrane, to
induce PAR-1 gene transcription. In this experiment, the luciferase
activity increased >2-fold in HMEC overexpressing CAAX-Raf
(Fig. 6A). As a known substrate for Raf kinase is the MAPK
kinase, MEK1, the upstream activator of MAPK, we also determined the
effects of overexpression of dominant negative MEK1. These results
indicated that the dominant negative MEK1 abolished the
thrombin-induced PAR-1 gene expression (Fig. 6B). However,
the oncogenic MEK1 induced the PAR-1-driven luciferase activity
(1.8-fold over untreated) independent of thrombin stimulation, a
response similar to the constitutively active mutants of Ras and Raf.
Western blot analysis using an anti-hemagglutinin polyclonal antibody
demonstrated the relative expression of mutant MEK1 isoforms in HMEC
(Fig. 6B, inset). Thus, the Ras/Raf/MAPK
signaling pathway is critical in mediating the thrombin-induced
expression of PAR-1 gene in endothelial cells.
Inhibition of Protein-tyrosine Kinase Signaling Pathways Prevents
Thrombin-induced PAR-1 Gene Expression--
Because tyrosine kinase
signaling activates Ras (32), we assessed the role of protein-tyrosine
kinase (PTK)-dependent signaling in mediating PAR-1 gene
expression in HMEC. Genistein, a PTK inhibitor, was used to investigate
PTK-dependent signal transduction. Thrombin-activated MAPK
phosphorylation was measured after pretreating HMEC with 30, 100, or
300 µM genistein. We showed that genistein decreased the
thrombin-induced MAPK phosphorylation in a dose-dependent manner (Fig. 7A).
To assess the role of PTK-dependent signaling in PAR-1 gene
transcription, HMEC transfected with luciferase-reporter plasmids were
pretreated with 100 µM genistein for 2 h prior to
stimulation with 100 nM thrombin. These results showed that
genistein pretreatment inhibited luciferase activity by 80% (Fig.
7B), whereas basal luciferase activity was unaffected by PTK
inhibition. Genistein also prevented the thrombin-induced increase in
PAR-1 mRNA levels at 3 and 6 h (Fig. 7C),
indicating the dependence of PTK signaling in the induction of PAR-1 gene.
Following functional depletion of endothelial cell PAR-1, cellular
resensitization to thrombin required 18 h and the response was
de novo PAR-1 synthesis-dependent (5).
Therefore, we examined a functional consequence of genistein on the
protein synthesis-dependent resensitization to thrombin at
18 h. HPAEC were pretreated with 100 µM genistein
prior to measuring thrombin-induced cell shape change by the electrical
resistance method (25) (see "Experimental Procedures"). The drop in
transendothelial electrical resistance (a measure of cell retraction)
in response to the initial thrombin concentration, which was sufficient
to deplete PAR-1 in HPAEC, was genistein-insensitive (Fig.
8, inset); however, the
protein synthesis-dependent cell retraction (i.e.
the cycloheximide-sensitive response) occurring at 18 h after
thrombin challenge (5) was inhibited 70% by genistein (Fig. 8).
Therefore, genistein did not prevent the initial thrombin-induced HPAEC
shape change, but it prevented the delayed, cycloheximide-sensitive
response following PAR-1 depletion.
Shc Phosphorylation following Thrombin Treatment--
The
activation of Ras by cell surface receptors involves formation of a
Shc-Grb2-SOS1 complex that depends on Src homology 2-mediated
recognition of phosphotyrosine (33). Src-mediated tyrosine
phosphorylation of Shc is essential for this complex formation (34). As
phosphorylation of Shc is critical in such protein-protein interactions
(35), we determined the phosphorylation of Shc adapter proteins in
HMEC. Phosphotyrosine-enriched proteins were immunoprecipitated from
lysates of thrombin-stimulated cells. Phosphorylation of p46, p52, and
p66 Shc as detected by anti-Shc immunoblotting increased following 1-2
min of thrombin exposure (Fig.
9A). Pretreatment of HMEC with
Src kinase inhibitor PP1 markedly reduced the thrombin-induced tyrosine
phosphorylation of Shc isoforms. Fig. 9B shows the
expression of all three isoforms of Shc in HMEC. These results indicate
that Src-mediated Shc phosphorylation serves as an upstream signaling
intermediate contributing to thrombin-activated Src homology
2-dependent protein interactions. Inhibition of Src Kinase Activity Prevents Thrombin-induced MAPK Phosphorylation and
Luciferase Expression Phosphoinositide 3-Kinase Activity Is Necessary for
Thrombin-induced PAR-1 Gene Expression--
PI3Ks, lipid kinases
existing in heterodimeric (PI3K Irreversible activation and degradation of PAR-1 precludes
receptor recycling as a means of restoring responsivity to thrombin via
the cell surface PAR-1 (10). As PAR-1 is cleaved and targeted to
lysosomes for degradation (10), it is necessary that PAR-1 be
re-synthesized to replenish functional cell surface receptor. Although
endothelial cells contain a preformed cytosolic pool of PAR-1 that
translocates within minutes to the cell surface in response thrombin
(10, 11), this response is independent of de novo receptor
synthesis and is rapidly lost once the pre-formed PAR-1 pool has been
depleted (5, 10). Inhibition of protein synthesis by cycloheximide
prevented the full recovery of cell surface PAR-1 after
thrombin-induced PAR-1 depletion (5), indicating de novo
protein synthesis was required for the response. In the present study,
we demonstrate that activation of cell surface PAR-1 by thrombin is
itself a critical stimulus for PAR-1 gene expression in vascular
endothelial cells. We showed in HPAEC and HMEC that PAR-1 activation
increased PAR-1 mRNA and PAR-1 promoter-driven luciferase-reporter
activity. Activation of PAR-1 was sufficient to increase PAR-1 mRNA
since endothelial cells challenged with PAR-1 activating peptide
(TRP-14), also induced PAR-1 gene expression. Therefore, the protein
synthesis-dependent PAR-1 resensitization in vascular
endothelial cells is attributed to the activation of PAR-1 gene expression.
Studies on the effects of thrombin on PAR-1 mRNA in human
glomerular mesangial and human erythroleukemia (HEL) cells (41, 42)
showed that PAR-1 mRNA was unaffected by thrombin in mesangial cells, whereas PAR-1 mRNA increased within 6 h of thrombin
exposure in HEL cells. HEL cells undergo prolonged PAR-1
desensitization following thrombin exposure (43); however, HEL cells,
unlike endothelial cells, lack a cytosolic PAR-1 pool (43). Recovery of
thrombin sensitivity in HEL cells was associated with new receptor synthesis since cycloheximide prevented the resensitization (43). Results of the present study showing that thrombin activation of PAR-1
in endothelial cells induced PAR-1 gene expression within 3-6 h after
thrombin exposure are consistent with the HEL cell experiments
(43).
Proteolytic cleavage of the surface receptors PAR-1, PAR-2, PAR-3, and
PAR-4 by proteases (i.e. thrombin in the case of PAR-1, PAR-3, and PAR-4 and tryptase or trypsin in the case of PAR-2) unmasks
a cryptic ligand that in PAR-1 interacts with the extracellular loop 2 (12, 44-47). Binding of the tethered ligand to extracellular loop 2 domain stimulates PAR-1, and generates intracellular second messengers
(47). PAR-2 and PAR-3 have been shown to be expressed in human
umbilical vein endothelial cells (48, 49). PAR-2 is trypsin-specific
receptor, but it can also be activated by PAR-1 activating peptide
(TRP-14) (50). PAR-3 and PAR-4 cannot be activated by TRP-14 (45, 46).
In the present study, we were unable to show a functionally active
PAR-2 in HPAEC since the PAR-2-specific activating peptide (SLIGKV) did
not induce either cell retraction or increase in
[Ca2+]i (data not shown).
Therefore, the activation of TRP-14-induced PAR-1 gene expression can
only be ascribed to stimulation of PAR-1.
Thrombin activates signaling in endothelial cells by coupling to PAR-1
to heterotrimeric Gi and Gq (13, 51). Our
results using PTX to address the role of heterotrimeric Gi
in activating PAR-1 gene showed that thrombin-induced PAR-1 gene
transcription was PTX-sensitive. To explore further the role of
PTX-sensitive heterotrimeric G proteins, a minigene encoding a peptide
antagonist homologous to the G We measured the activation of MAPK to ascertain MAPK involvement in the
signal transduction pathway downstream of Gi.
Phosphorylation of MAPK on threonine and tyrosine residues by MEK is
known to stimulate MAPK activity (52). We showed that thrombin
maximally activated MAPK phosphorylation within 2 min, and pretreatment of HMEC with PTX prevented the thrombin-induced MAPK phosphorylation. PTX also abolished MAPK-dependent phosphorylation of Elk-1
in an in vitro kinase assay. To delineate the signaling
mechanisms conveying the thrombin signal to PAR-1 promoter, we
introduced dominant negative (dn) mutants of Ras and MEK1 into HMEC. We
showed that overexpressing dn-mutant of Ras or MEK1 interfered with
activation of downstream signaling. Expression of dn-Ras (N17Ras) in
contrast to dn-MEK1 inhibited PAR-1 gene induction by 50% in response
to thrombin challenge. This may be the result of incomplete inhibition of thrombin-mediated Ras activation by N17 Ras expression or
alternatively to a Ras-independent signaling mechanism (53). The
finding that dn-MEK1 mutant fully inhibited PAR-1 gene transcription
following thrombin stimulation implies that MAPK activity is necessary
for PAR-1 gene induction since MEK1 is situated directly upstream of
MAPK in the Ras/Raf/MAPK signaling cascade. We also showed that
overexpressing the constitutively active mutants of Ras (V12Ras), Raf
(CAAX-Raf), and MEK1 (S218E/S222E) resulted in
transactivation of the PAR-1 promoter independent of thrombin stimulation.
Genistein, as in the case of PTX, prevents thrombin-induced
p21ras and MAPK activity (22), suggesting the
involvement of protein-tyrosine kinase (PTK) activity in the
Gi-transduced signaling of PAR-1 gene expression. In
thrombin-stimulated endothelial cells, we showed that genistein
pretreatment prevented MAPK phosphorylation and PAR-1 gene
induction, as demonstrated by both reporter assay and mRNA
blotting. Genistein has been shown to prevent the de novo
protein synthesis-dependent resensitization to thrombin
following PAR-1 depletion in endothelial cells (5). Thus,
thrombin-induced activation of PAR-1 promoter relies on the
PTK-dependent signaling pathway.
PI3K and Src-like tyrosine kinases lie between Gi and the
downstream Ras/MAPK signaling cascade (14, 15). We showed that inhibition of Src activity by PP1 prevented the induction of PAR-1 gene
in response to thrombin. In addition, overexpression of
dn-Src abrogated PAR-1 gene induction, whereas wild type Src
had no effect on thrombin signaling. Previous studies have linked
Src-like PTK activity to the formation of Shc-Grb2-SOS1, which in turn
activates p21ras (14, 15), and phosphorylation
of Shc on tyrosine residues by Src kinase is a prerequisite for Grb2
association and subsequent SOS1 recruitment (34). We showed that
thrombin stimulated the rapid tyrosine phosphorylation of Shc adapter
proteins and that this was inhibited by Src kinase inhibitor PP1,
suggesting that Src can activate Ras/MAPK via this mechanism, and thus
signal PAR-1 expression.
Thrombin-induced MAPK phosphorylation and PAR-1 gene expression were
sensitive to PI3K inhibition by both wortmannin and LY294002. The p85
subunit of PI3K In summary, the present data indicate that thrombin-mediated PAR-1 gene
expression in endothelial cells requires the heterotrimeric Gi-activated Ras/MAPK signaling pathway. The pathway
linking Gi to Ras relies on PI3K and Src-like PTK activity
as well as Shc-Grb2-SOS1 complex formation. We propose that
thrombin-induced PAR-1 gene expression activates the
Gi-linked Ras/MAPK cascade resulting in transactivation of
the PAR-1 promoter and PAR-1 expression.
i prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented
thrombin-induced MAPK phosphorylation, indicating a role of
G
i in activating the downstream MAPK pathway. Expression
of constitutively active G
i2 mutant or G
1
2
subunits increased reporter activity 3-4-fold in the absence of
thrombin stimulation. Co-expression of dominant negative mutants of
either Ras or MEK1 with the reporter construct inhibited the
thrombin-induced PAR-1 expression, whereas constitutively active forms
of either Ras or MEK1 activated PAR-1 expression in the absence of
thrombin stimulation. Expression of dominant negative Src kinase or
inhibitors of phosphoinositide 3-kinase also prevented the MAPK
activation and PAR-1 expression. We conclude that thrombin-induced
activation of PAR-1 mediates PAR-1 expression by signaling through
Gi1/2 coupled to Src and phosphoinositide 3-kinase, and
thereby activating the downstream Ras/MAPK cascade.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i2 (Q205L in
pcDNA1), G
1 (in pcDNA3.1), G
2 (in pcDNA3.1), bovine
GRK2 (in pcDNA3.0), and
subunit of bovine retinal transducin
(in pcDNA1.0) were from Dr. T. Voyno-Yasenetskaya (University of
Illinois, Chicago, IL). These expression constructs were prepared as
described (21). Expression plasmids encoding Gi2 minigene
antagonists (coding sequence 5'-ATCAAGAACCTGAAGGACTGCGGCCTTC-3' in
pcDNA3.1), and minigene with scrambled sequence (coding sequence
5'-ATGGGAAACGGCATCAAGTGCCTCTTCAACGACAAGCTG-3'in pcDNA3.1)
were from Dr. H. E. Hamm (Northwestern University, Chicago, IL).
The complimentary oligonucleotides were synthesized and annealed at
65 °C, and the annealed double-strand DNA was ligated in
pcDNA3.1 vector (23).2
The wild-type c-Src and dominant negative mutant c-Src (K295M/Y527F) in
pSM vector were obtained from Dr. S. Gutkind (NIDR, National Institutes
of Health, Bethesda, MD). Ras mutants cloned into pMAMneo expression
vector were from Dr. P. Sass (University of Illinois, Chicago, IL).
Electrodes for endothelial cell monolayer resistance measurements were
purchased from Applied Biophysics, Inc., Troy, NY.
70 °C. After centrifugation, the RNA
pellet was collected, washed with 75% ethanol, dried, and dissolved in
diethyl pyrocarbonate-treated water containing 0.5% SDS. RNA was
quantified by spectrophotometry. Approximately equal amounts of total
cellular RNA was separated on a denaturing 1% formaldehyde-agarose
gel. After separation, the RNA was transferred to Duralose membranes
and immobilized. The membranes were prehybridized for 1 h at
68 °C in Quickhyb solution and hybridized for 2 h at 68 °C
with a 1.36-kb thrombin receptor cDNA probe (provided by Dr.
S. R. Coughlin, University of California, San Francisco) and
1.1-kb glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The cDNA
probes were labeled with [32P]dCTP using a Prime-It II
kit (Stratagene). The membranes were washed and visualized by
autoradiography. Autoradiograms were scanned into a personal computer,
and levels of PAR-1 and GAPDH mRNA were quantified by NIH Image
version 1.6. -Fold increase was determined as PAR-1/GAPDH ratio) × (untreated PAR-1/GAPDH ratio)
1.
-glycerol
phosphate, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml
aprotinin, and 44 µg/ml PMSF) for 10 min at 4 °C. Cells were
scraped from dishes, and lysates were transferred to microcentrifuge
tubes and incubated 4 °C for 30 min. Insoluble material was removed
by centrifugation at 13,000 × g for 15 min, and
precleared supernatants were used for immunoprecipitation and in
vitro kinase assay according to the manufacturer's instructions.
Briefly, lysates were immunoprecipitated with phosphospecific p42/44
MAPK monoclonal antibody at 4 °C for 1 h. Protein A-Sepharose
beads were added and incubated at 4 °C for 2-4 h with gentle
rotation. Immunoprecipitates were pelleted, washed twice in lysis
buffer, and twice in kinase assay buffer (25 mM Tris-HCl,
pH 7.5, 1 mM Na3VO4, 1 mM
-glycerol phosphate, 2 mM dithiothreitol,
10 mM MgCl2). Immune complexes were resuspended in 50 µl of kinase assay buffer containing 200 µM ATP
and 2 µg of Elk-1 fusion protein and incubated at 30 °C for 30 min. Reactions were terminated by addition of 30 µl of SDS sample
buffer. Samples were resolved on 12% SDS-polyacrylamide gel,
transferred to Duralose membrane, and subsequently incubated with
phospho-Elk-1 antibodies. Phosphorylated Elk-1 was determined by the
ECL (enhanced chemiluminescence) method.
4 cm2). Prior to experiments,
monolayers were washed two times with serum-free medium and incubated
for 2 h in 1% serum-supplemented culture medium. The small and
large electrodes were connected to a phase-sensitive lock-in amplifier.
A constant current of 1 µA was applied by a 1-V, 4000-Hz AC connected
serially to a 1-megohm resistor between the small electrode and the
larger counter electrode. The voltage change between small electrode
and larger counter electrode was continuously monitored by lock-in
amplifier, stored, and processed on a computer. The data are presented
as change in resistive (in-phase) portion of the impedance normalized to its initial value at time zero as described (25, 26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Thrombin induces PAR-1 gene
transcription. Endothelial cells grown to confluence were
stimulated with thrombin or TRP-14 for the indicated times prior to
isolation of total cellular RNA and Northern blot analysis (described
under "Experimental Procedures"). A, HPAEC stimulated
with 25 nM thrombin. B, HMEC stimulated with 100 nM thrombin. C, HPAEC stimulated with 25 mM TRP-14. Band intensity was quantified using NIH Image
version 1.6, and ratios of PAR-1 to GAPDH plotted for each
autoradiogram are shown. Results are representative of two to five
experiments per group.
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Fig. 2.
PAR-1-driven luciferase expression by
activation of PAR-1. HMEC grown in six-well plates, transfected
with the PAR-1/Luciferase (300 ng) and TK/pRL (20 ng) reporter
plasmids, were stimulated with 100 nM thrombin or 100 mM TRP-14 and lysed at indicated times. Firefly and sea
pansy luciferase activity was measured from 20 µl of total cell
lysate (described under "Experimental Procedures"). Luciferase
reporter activity (relative light unit ratio (RLU)/mg of protein) was
expressed after subtracting the basal activity at each time point.
Black columns, 100 nM thrombin
treatment; white columns, 100 mM
TRP-14 treatment. Time course where each point (shown as mean ± S.E.) represents three to six wells per experiment. Experiments were
performed four times.
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Fig. 3.
Thrombin-mediated induction of PAR-1-driven
luciferase activity is pertussis toxin-sensitive. HMEC were
transfected with luciferase-reporter plasmids (described under
"Experimental Procedures") and pretreated overnight with pertussis
toxin (100 ng/ml). HMEC were then stimulated with 100 nM
thrombin for 4 h and lysed, and luciferase activity was measured.
RLU were corrected for milligrams of total protein. Luciferase activity
(mean ± S.E.) from HMEC -fold increase is [(RLU/mg protein
treated)/(RLU/mg protein untreated)]. Black
columns, 100 nM thrombin treatment;
white columns, without thrombin treatment. The
mean ± S.E. from four experiments repeated in triplicate are
shown. Asterisk (*) indicates the difference from
thrombin-stimulated control (p < 0.005).
subunits of heterotrimeric G proteins
and cytosolic domains of heptahelical receptors are potential targets
for interrupting agonist-mediated signaling events (23).2
To address further the role of Gi, we determined whether a
G
i-peptide antagonist interferes with thrombin-mediated
PAR-1 expression. We transfected plasmids encoding a peptide
corresponding to the COOH terminus of G
i to disrupt the
G protein-receptor interaction (23).2 Expression of the
G
i antagonist peptide inhibited thrombin-induced PAR-1
expression (Fig. 4A).
Overexpression of a scrambled peptide of the same amino acid
composition did not alter luciferase expression following thrombin
treatment. These results combined with the inhibitory effect of PTX on
PAR-1 gene expression indicate the involvement of Gi-PAR-1
coupling in mediating thrombin-induced PAR-1 gene induction.
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Fig. 4.
A, overexpression of peptide antagonists
specific for G i-receptor interface prevents
thrombin-induced increase in PAR-1 expression. HMEC were co-transfected
with the reporter plasmids and 100 ng of pCDNA 3.1 (vector),
G-scrambled minigene (G
scr), or G
i
minigene (G
i). Cells were stimulated with 100 nM thrombin for 4 h, lysed, and assayed for luciferase activity. Black
columns, 4 h of 100 nM thrombin treatment;
white columns, without thrombin treatment.
Results are mean ± S.E. from five experiments.
Asterisk (*) indicates the difference from
thrombin-stimulated control (p < 0.005). B,
constitutively active G
i2 mutant
(G
i2-Q205L) induces PAR-1 expression. HMEC were
co-transfected with the reporter plasmids (as described in Fig. 2) and
100 ng of G
i2-Q205L for 4 h. Cells were lysed and
assayed for luciferase activity. Black columns,
4 h of 100 nM thrombin treatment; white
columns, without thrombin treatment. Values are shown as
mean ± S.E. from four experiments made in triplicate.
Asterisk (*) indicates the difference from basal expression
(p < 0.001). C, G
expression in HMEC
activates PAR-1 expression. Plasmids encoding G
1 or G
2 were
co-transfected with the luciferase-reporter plasmids in HMEC.
Black columns, 4 h of 100 nM
thrombin treatment; white columns, without
thrombin treatment. Values are shown as mean ± S.E. from four
experiments of three wells in each group. Asterisk (*)
indicates difference from basal reporter activity (p < 0.005). D, G
scavenger co-expression prevents
thrombin-induced PAR-1 expression. Plasmids encoding GRK2 or transducin
were co-transfected with the luciferase-reporter plasmids in HMEC.
Black columns, 4 h of 100 nM
thrombin treatment; white columns, without
thrombin treatment. Values are shown as mean ± S.E. from four
experiments of three wells in each group.
and
subunit-dependent manner (14, 15, 27). To
address if
or
subunits coupling to PAR-1 is responsible for
downstream signaling, we co-expressed a constitutively active G
i2 mutant (Gi2-Q205L) or
subunits with
reporter construct in HMEC. Expression of mutant G
i2
increased reporter activity 3-4-fold over basal values (Fig.
4B), suggesting the involvement of PTX-sensitive G proteins
in the thrombin-induced PAR-1 gene induction. Co-expression of plasmids
encoding the G
1- and G
2-isoforms along with luciferase-reporter construct induced PAR-1 gene expression in the absence of thrombin treatment (Fig. 4C). However, neither G
1 nor G
2 alone
induced PAR-1 gene expression. Further, we co-expressed the G
sequesters such as G protein-coupled receptor kinase 2 (GRK2) and
transducin with the luciferase-reporter construct (28, 29).
Co-expression of either GRK2 or transducin with the PAR-1 reporter
construct markedly reduced thrombin-induced PAR-1 expression (Fig.
4D). These results indicate that G
i
activation mediates thrombin-induced PAR-1 expression by G
dissociation from the ligand-activated G
i.
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Fig. 5.
Thrombin-induced MAPK phosphorylation is PTX
sensitive. Confluent HMEC pretreated with 100 ng/ml PTX for
20 h were stimulated with thrombin prior to lysis. Approximately
30 µg of total cellular protein was resolved by SDS-PAGE, and Western
blot analysis was performed using phosphospecific MAPK antibodies (see
"Experimental Procedures"). A, time course of MAPK
phosphorylation induced by 100 nM thrombin. B,
PTX effect on thrombin-induced MAPK phosphorylation. For comparison,
cells were stimulated with 1.0 µM
12-O-tetradecanoylphorbol-13-acetate for 10 min where
indicated (B). Blots from A and B were
stripped and reprobed with anti-Erk1 antibodies. C, to
assess MAPK activity, phosphorylated MAPK was immunoprecipitated from
HMEC lysates (see "Experimental Procedures") and in
vitro kinase assay was performed using an Elk-1 GST fusion protein
as a MAPK substrate. Samples were resolved by SDS-PAGE and
immunoblotted with a phosphospecific Elk-1 polyclonal antibody. HMEC
were stimulated with 50 ng/ml basic fibroblast growth factor for 10 min
where indicated. Each immunoblot is representative of three
experiments.
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Fig. 6.
Blocking Ras/Raf/MAP kinase signal
transduction cascade prevents thrombin-induced PAR-1 expression.
HMEC were co-transfected with 100 ng of plasmid DNA encoding
(A) dominant-negative Ras (Ras S17N), constitutively active
Ras (Ras G12V), constitutively active Raf-1 (CAAX-Raf) or
(B) transfected with 300 ng of plasmid DNA encoding
dominant-negative MEK 1 (MKK1 K97M), constitutively active MEK 1 (MKK1S218E/S222E), and the luciferase-reporter plasmids. Cells were
grown to confluence, stimulated with thrombin (where indicated) for
4 h, lysed, and luciferase activity was measured. Black
columns, 4 h of 100 nM thrombin treatment;
white columns, without thrombin treatment.
Results are the mean ± S.E. from four experiments of three wells
from each group. Inset, lysates from HMEC, transfected with
increasing amounts of MKK1 plasmids were probed for MKK1 expression
using chicken anti-HA antibodies. Each MKK1 plasmid encodes MKK1-HA
chimera. In A, asterisk (*) indicates the
significant difference from thrombin stimulated control reporter
expression (p < 0.05). In B,
asterisk (*) indicates difference from basal reporter
expression (p < 0.05).
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Fig. 7.
A, genistein prevents thrombin-induced
MAPK phosphorylation. HMEC were pretreated with 30, 100, or 300 µM genistein for 2 h prior to 2 min stimulation with
100 nM thrombin. Approximately 30 µg of total cellular
protein was resolved by SDS-PAGE, and Western blot analysis was
performed using phosphospecific MAPK and anti-Erk1 antibodies (see
"Experimental Procedures"). Results are representative of three
experiments. B and C, genistein inhibits
thrombin-induced PAR-1 gene expression. B, HMEC were
transfected with the luciferase-reporter plasmids (as described in Fig.
2). Cells were pretreated with 100 µM genistein for
2 h prior to 4 h stimulation with 100 nM
thrombin. Cells were lysed, and luciferase activity was measured.
Values are shown as mean ± S.E. from four experiments of three
wells. Black columns, 4 h of 100 nM thrombin treatment; white columns,
without thrombin treatment. Asterisk (*) indicates
difference from thrombin-stimulated PAR-1 expression (p < 0.05). C, HPAEC were pretreated with 100 µM
genistein (or vehicle; 0.05% dimethyl sulfoxide) 2 h prior to
thrombin stimulation (25 nM) for indicated times. Total RNA
was isolated, and Northern blot analysis was performed. Band
intensities were quantified as described in Fig. 1. Ratios of PAR-1 to
GAPDH plotted for each autoradiogram are shown. The results shown are
mean of three experiments.
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Fig. 8.
Genistein prevents the delayed protein
synthesis-dependent thrombin-induced endothelial cell shape
change. HPAEC grown to confluence on gold electrodes were
pretreated for 2 h with 100 µM genistein (or
vehicle). Change in normalized electrical resistance following addition
of 25 nM thrombin at 0 h to genistein- or
vehicle-pretreated HPAEC was measured. Inset, after 4 h
of thrombin treatment, cells were washed three times and incubated in
complete medium containing 10% serum (no genistein or thrombin) until
the 16-h time point. Cells were washed and incubated in serum-free
medium without drug for 2 h prior to the subsequent, 25 nM thrombin challenge at 18 h. Results shown are from
a representative experiment.
Src kinase is an upstream regulator of Ras (36),
and stimulation of Src kinases by G protein-coupled receptors results
in tyrosine phosphorylation of Shc and subsequent p21ras activation (34, 37). We used two distinct
inhibitors, herbimycin A and PP1, to assess the role of Src family of
PTK in thrombin-induced intracellular signaling. Phosphorylation of
MAPK in response to 2 min of thrombin exposure was measured in HMEC
pretreated with 1, 10, or 30 nM herbimycin A (Fig.
10A). Herbimycin A
concentrations as low as 1 nM inhibited thrombin-induced
MAPK phosphorylation to levels comparable to unstimulated cells. PP1
(10 µM) also inhibited the thrombin-induced MAPK
phosphorylation (data not shown). In addition, pretreatment of HMEC
with 10 µM PP1 prevented thrombin-mediated increases in
luciferase activity (Fig. 10B). Moreover, co-expression of
dominant negative Src (c-Src K295M/Y527F) with the reporter plasmid
prevented the induction of luciferase expression (Fig. 11A). Western blot analysis
demonstrated the relative expression of c-Src in each group (Fig.
11B). Thus, Src kinase phosphorylates Shc to activate
downstream signals, and signals the thrombin-induced PAR-1 gene
expression.
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Fig. 9.
Thrombin stimulates tyrosine phosphorylation
of Shc by activation of Src kinase. A,
HMEC were preicubated with or without 10 µM PP1 for
2 h and then stimulated with 100 nM thrombin for 1 and
2 min before being lysed. Total cellular protein was incubated with
anti-phosphotyrosine (phospho-Y) monoclonal antibody. Other
experimental details are described under "Experimental Procedures."
Precipitated proteins were resolved by SDS-PAGE, transferred to
Duralose membranes, and probed with anti-Shc antibody. Phosphorylation
of p46, p52, and p66 Shc isoforms was detected in anti-phosphotyrosine
immunoprecipitates. B, HMEC lysates were resolved by
SDS-PAGE and anti-Shc Western blot was performed. +, Shc positive
control; IP, immunoprecipitation; IB,
immunoblot.
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Fig. 10.
Src family inhibitors prevent
thrombin-induced MAPK activation and PAR-1 gene expression.
A, HMEC pretreated for 2 h with herbimycin A (1, 10, and 30 nM) were assayed for MAPK phosphorylation following
2 min of 100 nM thrombin. Experiment was repeated twice
with similar results. B, HMEC transfected with
luciferase-reporter constructs were pretreated with 10 µM
PP1 2 h prior to 4 h of thrombin exposure. Cells were lysed,
and luciferase activity was measured. Black
columns, 4 h of 100 nM thrombin treatment;
white columns, without thrombin treatment. Data represent
mean ± S.E. of four experiments of three wells in each group.
Asterisk (*) indicates difference from the
thrombin-stimulated control (p < 0.05).
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Fig. 11.
Dominant-negative c-Src abolishes
thrombin-induced PAR-1 promoter-driven luciferase expression. The
luciferase-reporter plasmids were cotransfected with 100-300 ng of
plasmid DNA encoding dominant-negative c-Src (K295M/Y527F) or wild-type
(wt) c-Src for 2-4 h. After 24 h, HMEC were stimulated with 100 nM thrombin for 4 h. A, cells were lysed,
and luciferase activity was measured. Black
columns, 4 h of 100 nM thrombin treatment;
white columns, without thrombin treatment. Values
are shown as mean ± S.E. from four experiments of three wells
each group. Asterisk (*) indicates difference from thrombin-
stimulated control (p < 0.05). B, aliquots
from reporter assay lysates were resolved by SDS-PAGE and analyzed for
Src overexpression by anti-Src immunoblotting.
) and monomeric (PI3K
) forms, have
been implicated in G protein-coupled receptor-mediated signaling (38,
39). One isoform, PI3K
, is stimulated by G
derived from
ligand-activated G protein-coupled receptor (34, 37-40). This
activation recruits PI3K
to plasma membrane, which enhances
Src-like kinase activity and facilitates activation of
Shc-Grb2-Sos-Ras pathway (39, 40). We used pharmacologically distinct
inhibitors, wortmannin and LY294002, to address the role of PI3K in
thrombin-mediated signaling. Both inhibitors prevented thrombin-induced
MAPK phosphorylation at 2 min of thrombin stimulation (Fig.
12A). Moreover, wortmannin
(100 nM) and LY294002 (100 nM) pretreatment
inhibited thrombin-induced increase in reporter activity (Fig.
12B), indicating the involvement of PI3K in PAR-1 gene
induction. Thus, the PI3K activity upstream of MAPK is necessary for
thrombin-induced signaling of PAR-1 gene expression.
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Fig. 12.
A, wortmannin and LY294002 inhibit
thrombin-induced MAPK phosphorylation. HMEC pretreated for 2 h
with wortmannin (30, 100, and 300 nM) (upper) or
LY294002 (30, 100, and 300 nM) (lower) were
assayed for MAPK phosphorylation following 2 min of 100 nM
thrombin stimulation. Blots were stripped and reprobed with anti-Erk1
antibodies. Phospho-MAPK positive control (+). B, PI3K
activity is required for thrombin-induced PAR-1 gene expression. HMEC
transfected with luciferase-reporter constructs were pretreated with
100 nM wortmannin or 100 nM LY294002 for 2 h prior to 4 h of thrombin exposure. Cells were lysed, and
luciferase activity was measured. Black columns,
4 h of 100 nM thrombin treatment; white
columns, without thrombin treatment. Values are shown as
mean ± S.E. from four experiments of three to six wells in each
group. Asterisk (*) indicates difference from the thrombin-
stimulated control (p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COOH terminus of Gi was
co-expressed with the reporter construct in HMEC. We showed that
overexpression of this G
i peptide inhibited
thrombin-induced PAR-1 gene expression. Expression of constitutively
active mutant of G
i2 or G
subunits in HMEC was
sufficient to transactivate the PAR-1 promoter in absence of thrombin.
Further, we showed that co-expression of G
subunits sequesters
such as GRK2 and transducin with PAR-1 promoter construct prevented
thrombin-induced expression of PAR-1 in HMEC (28, 29). These data
demonstrate that G
i activation mediates thrombin-induced
PAR-1 expression by G
dissociation from the ligand-activated
G
i.
contains a Src homology 2 domain interacting with
Shc-Grb2 complex (39, 40), suggesting that activation of PI3K
can
lead to activation of the downstream PTK pathway, and thereby to PAR-1
gene expression. The present data are consistent with the hypothesis
that G
released from G
i activates Src-like PTK and
PI3K, and this in turn activates Ras/MAPK signal transduction, and
induces PAR-1 gene expression in endothelial cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL45638, GM58531, HL27016, T32HL07829, and GM56159.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: Cell and Cancer Biology Dept., Medicine Branch, DCS/NCI/NIH, Rockville, MD 20850-3300.
To whom correspondence and reprint requests should be
addressed: Dept. of Pharmacology (M/C 868), College of Medicine,
University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612-7343. Tel.: 312-355-0249; Fax: 312-413-0222; E-mail: tiruc{at}uic.edu.
2 Gilchrist, A., Buenemann, M., Li, A., Hosey, M. M., and Hamm, H. E., (1999) J. Biol. Chem. 274, 6610-6616.
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ABBREVIATIONS |
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The abbreviations used are: PAR-1, proteinase-activated receptor-1; HMEC, human dermal microvessel endothelial cell(s); HPAEC, human pulmonary artery endothelial cell(s); PTX, pertussis toxin; MAPK, mitogen-activated protein kinase; PTK, protein-tyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; kb, kilobase pair(s); HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PMSF, phenylmethylsulfonyl fluoride; PI3K, phosphoinositide 3-kinase; dn, dominant negative; MEK, MAPK kinase; PAGE, polyacrylamide gel electrophoresis; HEL, human erythroleukemia; RLU, relative light unit ratio.
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