Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK
involves multiple signaling pathways
Hong
Wang,
Joachim J.
Ubl,
Rolf
Stricker, and
Georg
Reiser
Otto-von-Guericke-Universität Magdeburg, Medizinische
Fakultät, Institut für Neurobiochemie, 39120 Magdeburg,
Germany
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ABSTRACT |
Protease-activated receptors (PARs), newly
identified members of G protein-coupled receptors, are widely
distributed in the brain. Thrombin evokes multiple cellular responses
in a large variety of cells by activating PAR-1, -3, and -4. In
cultured rat astrocytes we investigated the signaling pathway of
thrombin- and PAR-activating peptide (PAR-AP)-induced cell
proliferation. Our results show that PAR activation stimulates
proliferation of astrocytes through the ERK pathway. Thrombin
stimulates ERK1/2 phosphorylation in a time- and
concentration-dependent manner. This effect can be fully mimicked by a
specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP.
PAR-2-AP can induce a moderate ERK1/2 activation as well.
Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via
two branches: 1) the PTX-sensitive G
protein/(
-subunits)-phosphatidylinositol 3-kinase branch, and
2) the Gq-PLC-(InsP3
receptor)/Ca2+-PKC pathway. Thrombin- or PAR-1-AP-induced
ERK activation is partially blocked by a selective EGF receptor
inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor
is unlikely for ERK1/2 activation and is certainly not involved in
PAR-1-induced proliferation. The metalloproteinase mechanism involving
transactivation of the EGF receptor by released heparin-binding EGF was
excluded. EGF receptor activation was detected by the receptor
autophosphorylation site, tyrosine 1068. Our data suggest that
thrombin-induced mitogenic action in astrocytes occurs independently of
EGF receptor transphosphorylation.
protease-activated receptors; extracellular signal-regulated
protein kinase; calcium signaling; epidermal growth factor receptor; transactivation; mitogen-activated protein kinase
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INTRODUCTION |
IN ADDITION TO BEING A
PROTEASE involved in blood coagulation and tissue repair,
thrombin has also been shown to act as a multifunctional signaling
molecule, even in the brain (19). In astrocytes, thrombin
has been found to induce morphological changes, proliferation, and
secretion of endothelin-1 (13, 18). These
thrombin-stimulated cellular events are mediated through the
proteolytic activation of a seven-transmembrane domain G
protein-coupled receptor (GPCR) (5, 19), the so-called
protease-activated receptor (PAR). Activation of PARs is achieved when
the extracellular NH2 terminus of the receptor is cleaved
by the specific protease. The newly generated NH2 terminus
binds like a tethered ligand intramolecularly to extracellular loop 2 of the receptor (34), leading to the G protein-coupled
signal transduction, i.e., activation of phospholipase C (PLC),
generation of inositol 1,4,5-trisphosphate (InsP3),
increase of intracellular Ca2+, and activation of protein
kinase C (PKC). Synthetic peptides mimicking the sequence of the
tethered ligand can bind to and activate the receptor, bypassing the
requirement of proteolysis. Thrombin can activate PAR-1, -3, and -4 of
the PAR family, whereas PAR-2 is mainly activated by trypsin
(38).
Activation of mitogen-activated protein kinases (MAPKs) that comprise
the extracelluar signal-regulated protein kinase ERK1 (p44 MAPK) and
ERK2 (p42 MAPK) plays a crucial role in regulating cellular
proliferation and differentiation signals from the cell surface to the
nucleus (39). The initial characterization of the
activation mechanisms of MAPKs by cell surface receptors was revealed
by analysis of classic tyrosine kinase receptors such as the epidermal
growth factor (EGF) receptor (15). Multiple subsequent
studies showed that stimulation of many GPCRs also leads to rapid
activation of the ERK pathway (12). Most recently published data suggest that part of the mitogenic stimulus of some
GPCRs can be produced by transactivation of EGF receptor (8, 9,
45). The transactivation mechanism was found to be due to
release of soluble EGF receptor ligand upon stimulation of GPCRs by
thrombin, lysophosphatidic acid (LPA), endothelin, and carbachol
(21, 46).
Previous work in our laboratory has shown that all four different types
of PARs known so far are widely expressed in the brain (54). We have also demonstrated that rat astrocytes
functionally coexpress these four subtypes of PARs (62).
Short-term application of agonists for PAR-1 through -4 induces
increase in intracellular Ca2+. Furthermore, we found that
stimulation of PAR-1 and PAR-2 leads to proliferation of astrocytes
(62). MAPKs are activated by thrombin, leading to
proliferation in various cell types (36, 44, 56). Although
thrombin has been shown to activate MAPK in astrocytes
(4), the nature of the biochemical link from thrombin
receptors to MAPKs in astrocytes remains to be delineated. In the
present study we examined whether these PAR-evoked mitogenic signals
are transmitted through classic G protein-coupled signaling pathways or
cross-communication with EGF receptor. Experiments were performed to
identify the relationship between cell proliferation and activation of
ERK1/2 by using several pharmacological tools.
The novel mechanism implying activation of EGF receptor indirectly by
GPCRs potentially also provides new directions for clinical applications. Thrombin and PARs are targets for possible therapeutic interventions to induce neuroprotection. For possible treatment of
neurodegenerative diseases, it is highly important to understand whether the transactivation pathway is connected to PARs in brain cells, because PAR-1 activation appears to be able to promote neuronal
survival after ischemia (53) or brain trauma
(63). The main finding of this study is that activation of
PARs stimulates proliferation of rat cultured astrocytes via the
ERK/MAPK pathway. This involves two branches. The first branch goes
through PLC-InsP3/Ca2+-PKC and converges with
the second pathway, which comes from pertussis toxin (PTX)-sensitive G
proteins and phosphatidylinositol (PI) 3-kinase. There is, however, no
transphosphorylation of EGF receptor, occurring at autophosphorylation
site tyrosine 1068.
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MATERIALS AND METHODS |
Materials.
Human thrombin and EGF were from Sigma (St. Louis, MO). The synthetic
thrombin receptor agonist peptide (TRag;
Ala-parafluorPhe-Arg-Cha-homoArg-Tyr-NH2) and rat
PAR-2-activating peptide (PAR-2-AP) (SLIGRL,
H-Ser-Leu-Ile-Gly-Arg-Leu-NH2) were purchased from
Neosystem Laboratoire (Strasbourg, France). Human PAR-3-AP (TFRGAP,
H-Thr-Phe-Arg-Gly-Ala-Pro-OH) and rat PAR-4-AP (GYPGKF,
H-Gly-Tyr-Pro-Gly-Lys-Phe-OH) were purchased from Bachem (Heidelberg,
Germany). U-73343, U-73122, 2-aminoethoxydiphenylborate (2-APB),
PD-98059, AG1478, and wortmannin were purchased from Calbiochem (La
Jolla, CA); bisindolylmaleimide (GF-109203X) was from LC Laboratories
(Grünberg, Germany); and PTX was from Alexis (San Diego, CA).
Cell cultures.
Primary astrocyte-enriched cell cultures were obtained from two newborn
rats according to a previously published method (58). All
experiments conformed to guidelines from Sachsen-Anhalt on the ethical
use of animals, and all efforts were made to minimize the number of
animals used. In brief, newborn rats were decapitated, and total brains
were removed and collected in ice-cold Puck's-D1 solution composed of
(in mM) 137.0 NaCl, 5.4 KCl, 0.2 KH2PO4, 0.17 Na2HPO4, 5.0 glucose, and 58.4 sucrose, pH 7.4. The brains were gently passed through nylon mesh (136-µm pore width)
and centrifuged at 4°C for 5 min at 500 g. The cells were
resuspended in 10 ml of Dulbecco's modified Eagle's medium
supplemented with 10% (vol/vol) fetal calf serum, 20 U/ml penicillin,
and 20 µg/ml streptomycin (Biochrom, Berlin, Germany). The cells were
plated on round coverslips (22-mm diameter) placed in culture dishes (50-mm diameter) at a density of 2.5-5.0 × 105
cells/dish and incubated at 37°C with 10% CO2,
humidified to saturation. The medium was changed for the first time
after 5 days and thereafter every 2-3 days, depending on the cell
density. For experiments cells were used between days 7 and
14 in culture. The purity of astrocyte culture was
determined by immunofluorescence using a mouse monoclonal antibody
against glial fibrillary acidic protein (GFAP; Boehringer Mannheim,
Mannheim, Germany), an astrocyte-specific marker. Alexa 488 anti-mouse
IgG antibody (Molecular Probes, Eugene, OR) was used as the secondary
antibody. Confluent monolayers of astrocytes showed >97% positive
staining for GFAP.
Cytosolic Ca2+ measurement.
The free intracellular Ca2+ concentration
([Ca2+]i) was determined by using the
Ca2+-sensitive fluorescent dye fura 2. For dye loading, the
cells grown on a coverslip were removed from the culture dish and
placed in 1 ml of HEPES-buffered saline (HBS) for 30 min at 37°C,
supplemented with 2 µM fura 2-AM (Molecular Probes). HBS has the
following composition (in mM): 145 NaCl, 5.4 KCl, 1 MgCl2,
1.8 CaCl2, 25 glucose, and 20 HEPES, pH 7.4 adjusted with
Tris. Loaded cells were transferred into a perfusion chamber with a
bath volume of about 0.2 ml and mounted on an inverted microscope
(Axiovert 135; Zeiss, Jena, Germany). During the experiments the cells
were continuously superfused with buffer heated to 37°C.
Single-cell fluorescence measurements of
[Ca2+]i were performed by using an imaging
system from TILL Photonics (Munich, Germany). Cells were excited
alternately at 340 and 380 nm for 30-100 ms at each wavelength
with a rate of 0.33 Hz, and the resultant emission (F340
and F380) was collected above 510 nm. Images were stored on
a personal computer, and subsequently the changes in fluorescence ratio
(F340/F380) were determined from selected
regions of interest covering single astrocytes.
Proliferation assay.
Astrocytes were plated at a density of 2 × 103
cells/well in 96-well plates and were serum-starved for 24 h
before experiments. All experiments were carried out with a minimum of
six wells per condition (n
6) with at least two different
preparations. For assessing the proliferation 24 h later, we used
the CellTiter 96 AQueous One solution cell proliferation
assay (Promega, Madison, WI) in accordance with the manufacturer's
instructions. Absorption was measured at 490 nm with a microplate
reader (Molecular Devices). Proliferation is given as the percent
change compared with control. The proliferative effect induced by
thrombin, TRag, or PAR-2-AP was further confirmed by the measurement of
5-bromodeoxyuridine (BrdU) incorporation according to Yeh et al.
(64).
ERK1/2 phosphorylation.
Confluent cells were deprived of serum for 24 h before use, and
drug treatments were carried out at 37°C as indicated in
RESULTS. After stimulation, monolayers were washed twice
with ice-cold phosphate-buffered saline (PBS) and lysed in modified
RIPA buffer [50 mM Tris, pH 7.4, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM
Na3VO4, 1 mM NaF, and one tablet of protease
inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany)
per 50 ml]. The cell lysate was gently shaken on a rocker for 15 min
at 4°C. The lysate was centrifuged at 14,000 g in a
precooled centrifuge for 15 min, the supernatant was immediately transferred to a fresh centrifuge tube, and the pellet was discarded. Protein concentration was determined by the Bradford method using bovine serum albumin as standard. Samples containing equal amounts of
protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (20 µg/lane) and transferred to nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and rinsed in PBS with 0.1% Tween 20 3 times. Membranes were then incubated for 90 min at room temperature with specific antibodies against phosphorylated ERK1/2 [phospho-p44/42 MAPK
(Thr202/Tyr204; 1:2000)] or against ERK1/2
[p44/42 MAPK (1:2,000)] (New England Biolabs, Beverly, MA). After
three rinses, membranes were further incubated for 90 min at room
temperature with peroxidase-conjugated anti-mouse or anti-rabbit IgG
(1:10,000, respectively; Dianova, Hamburg, Germany). Membranes were
washed three times, and proteins were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech). Band intensity was
quantified by using a GS-800 calibrated densitometer (Bio-Rad) with
Quantity One quantitation software.
Immunoprecipitation and immunoblotting.
In the experiments for establishing possible EGF receptor activation,
stimulations were carried out at 37°C in serum-free medium. After
stimulation, monolayers in 60-mm culture dishes were washed twice with
ice-cold PBS and lysed in ice-cold modified RIPA buffer. The cell
lysate was treated as described in ERK1/2 phosphorylation. Protein (500 µg) was incubated with 5 µg of rabbit polyclonal antibody against the EGF receptor (New
England Biolabs) for 4 h at 4°C and then with protein
A-conjugated agarose beads overnight with constant shaking at 4°C.
Immune complexes were washed three times with ice-cold RIPA buffer,
denatured in Laemmli sample buffer, and resolved by 7.5% SDS-PAGE.
Tyrosine phosphorylation or the presence of immunoprecipitated proteins
was detected by protein immunoblotting. Phosphotyrosine was detected by
using a 1:500 dilution of anti-phosphotyrosine monoclonal antibody
clone 4G10 (Biomol). EGF receptor protein was detected by using a 1:500 dilution of rabbit polyclonal antibody against the EGF receptor.
Statistics.
Statistical evaluation was carried out using Student's
t-tests, and P < 0.05 was considered to be
significant. Data are given as means ± SE. All control values are
relative values calculated by dividing all single absolute values by
the mean of all control values (an absolute value) and then making a
group statistic of those relative values.
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RESULTS |
Activation of PARs stimulates ERK1/2 phosphorylation.
To examine whether stimulation of PARs can elicit ERK1/2 activation in
astrocytes, we challenged serum-starved astrocytes with thrombin (1 U/ml) for varying lengths of time, ranging from 5 min to 3 h. The
amount of phosphorylated ERK1/2 in astrocytes was determined by Western
blot analysis and was normalized by the total amount of ERK1/2. As
shown in Fig. 1, A and
B, thrombin can time-dependently induce ERK1/2
phosphorylation in astrocytes. The strongest activation was obtained at
5 min, which is consistent with other data (4). This
phosphorylation decreased gradually but persisted for up to 3 h.
In the following study, the phosphorylation of ERK1/2 is
expressed as a percentage of the phosphorylation of ERK1/2 seen after 5 min of stimulation with thrombin (10 U/ml) (see Fig. 2A).

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Fig. 1.
Time course of activation of extracellular
mitogen-regulated kinases ERK1/2 by thrombin in rat astrocytes.
Serum-starved astrocytes were exposed to thrombin (1 U/ml) for the time
period indicated. Phosphorylated ERK1/2 (pERK1 and pERK2) were detected
by Western blotting. A: representative blot from 1 experiment. Upper blot shows the phosphorylation of ERK1/2 induced by
thrombin; lower blot demonstrates equal loading of protein by detecting
total ERK1/2. B: bands were quantified by densitometer. The
amount of ERK1/2 phosphorylation was normalized by referring to the
total amount of ERK1/2. Here and in Fig. 2, the phosphorylation of
ERK1/2 is expressed as a percentage of the phosphorylation of ERK1/2
after 5 min of thrombin (10 U/ml) stimulation (see Fig. 2A).
Data represent means ± SE of 3 experiments.
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Fig. 2.
Concentration dependence of thrombin- and protease-activated
receptor-activating peptide (PAR-AP)-induced phosphorylation of ERK1/2
in rat astrocytes. Serum-starved astrocytes were stimulated by PAR
agonist for 5 min. Representative blots are shown for stimulation with
thrombin (0.01-10 U/ml) in A, PAR-1-AP (TRag;
0.01-10 µM) in B, PAR-2-AP (SLIGRL; 1-500 µM)
in C, PAR-3-AP (TFRGAP; 1-500 µM) in D,
and PAR-4-AP (GYPGKF; 1-500 µM) in E. F:
bands were quantified by densitometer and results are given as relative
values (see legend to Fig. 1). Data represent means ± SE of
3-5 experiments. *P < 0.05, **P < 0.01 vs. control.
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When astrocytes were treated with increasing concentrations of thrombin
(0.01-10 U/ml) for 5 min, ERK1/2 were concentration-dependently phosphorylated with the most pronounced effect at the concentration of
10 U/ml, as shown in Fig. 2, A
and F. Because thrombin activates not only PAR-1 but
also PAR-3 and -4, we had to differentiate between different PARs.
Therefore, we also applied the respective PAR-APs as stimulatory
agents. As shown in Fig. 2, B-F, the activating peptides for PAR-1, -2, -3, and -4 each evoke some ERK1/2 activation, but to a very different degree. Compared with the response induced by
10 U/ml thrombin, 95% was achieved by 10 µM thrombin receptor agonist (TRag), which is a potent (16) and specific
PAR-1-AP (28). Only 40% activation could be achieved by
PAR-2-AP (SLIGRL) at 500 µM. However, a negligible stimulation of 10 and 8% was elicited by PAR-3-AP and PAR-4-AP (500 µM TFRGAP and
GYPGKF), respectively. Compared with control cells, significant ERK1/2 phosphorylation was also observed with 0.1 and 1 µM TRag stimulation, 10-100 µM PAR-2 AP stimulation, and 100 µM PAR-3 AP
stimulation. PAR-3-AP (TFRGAP) was initially shown to be inactive
(26). It is intriguing that we found that this peptide
could elicit Ca2+ mobilization in astrocytes
(62). In addition, PAR-3-AP is also capable of stimulating
ERK1/2, as shown here. Our previous studies had indicated that this
peptide is most unlikely to signal through PAR-1 or PAR-2
(62) but, rather, genuinely activates PAR-3. Nevertheless,
caution is still needed to interpret these results. To clarify
unequivocally that PAR-3-AP is indeed activating PAR-3 will require
independent expression of rat PAR-3 against a null background and
examination of its signaling abilities.
Taken together, these results suggest that activation of PARs
stimulates ERK1/2 phosphorylation in astrocytes. However, not all PAR
subtypes contribute to the effects induced by thrombin stimulation. The
effect of thrombin is mediated mainly through PAR-1, because the effect
can almost completely be mimicked by TRag. This is consistent with our
recently reported data about proliferation induced by thrombin and TRag
in astrocytes (62), as also shown in Fig.
3C. Thus, within the PAR
system, PAR-1 seems to be the predominant receptor for the subsequent
cellular consequence of exposure of astrocytes to thrombin. Therefore,
in the following experiments the elucidation of the transduction
mechanism underlying the thrombin response focused on the signaling
cascades mediated through PAR-1.

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Fig. 3.
Effects of PD-98059 on thrombin- and TRag-evoked ERK1/2
phosphorylation and proliferation in rat astrocytes. Serum-starved
astrocytes were preincubated with PD-98059 (100 µM) for 15 min before
5 min of stimulation with thrombin (1 U/ml) or TRag (1 µM).
A: representative blot of ERK1/2 phosphorylation.
B: quantification by densitometer. ERK1/2 phosphorylation
induced by thrombin or TRag alone was taken as 100%. The same relative
quantification was used in Figs. 4-8. Data represent means ± SE from at least 3 experiments. **P < 0.01 vs. cells
exposed to thrombin or TRag alone. C: serum-starved
astrocytes were preincubated with PD-98059 (100 µM) for 15 min before
3 h of stimulation with thrombin (10 U/ml) or TRag (10 µM).
Hatched bars, values measured by BrdU incorporation assay. Open and
solid bars, values detected by CellTiter 96 AQueous One
solution cell proliferation assay. In Figs. 4-8, proliferation was
also assessed using the latter assay. Proliferation is expressed as
percent change compared with control. Data represent means ± SE
(n 6 wells/condition). *P < 0.05 vs. cells exposed to thrombin or TRag alone.
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Role of ERK/MAPK in the mitogenic process initiated by PAR-1
activation.
ERK1/2, which are believed to be a key component for the mitogenic
signal transduction, are phosphorylated as a result of PAR-1 activation
in a variety of cell types (14, 36, 56). Therefore,
PD-98059, a specific MAP kinase kinase (MEK) inhibitor, was applied in
the proliferation assays where astrocytes were treated with thrombin or
TRag. As shown in Fig. 3C, both thrombin (10 U/ml)- and TRag
(10 µM)-induced proliferation in astrocytes were totally blocked by
PD-98059 (100 µM). Interestingly, as shown in Fig. 3, A
and B, a 15-min preincubation with PD-98059 completely suppressed the ERK1/2 phosphorylation induced by thrombin (1 U/ml) or
TRag (1 µM) as well. These results indicate that the
proliferation-enhancing effect of thrombin and TRag was mediated
through ERK/MAPK activation.
Effect of PTX on thrombin-induced proliferation and ERK
phosphorylation.
PARs are GPCRs signaling via heterotrimeric G proteins. Thus the type
of G proteins involved in thrombin-induced
[Ca2+]i mobilization as well as proliferation
and ERK1/2 phosphorylation in astrocytes was studied by applying PTX.
PTX can inactivate the Go/Gi family but not
affect others. Cells were incubated with PTX (200 ng/ml) for 24 h
before stimulation.
After preincubation with PTX, the increase in
[Ca2+]i evoked by thrombin or TRag was
attenuated by 44 and 63%, respectively (Table
1). As Fig.
4C shows, pretreatment with
PTX also strongly inhibited the proliferative effects of thrombin and
TRag in astrocytes. Moreover, ERK1/2 phosphorylation by thrombin and
TRag was also partially diminished due to the
Go/Gi protein inactivation by PTX (Fig. 4,
A and B). This partial inhibition was not due to a nonspecific effect because EGF-stimulated ERK1/2 phosphorylation was
not affected by the PTX pretreatment (Fig. 4, A and
B). These results suggest that the signaling cascade from
PAR-1 to the ERK/MAPK is mediated through PTX-sensitive as well as
PTX-insensitive G proteins.

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Fig. 4.
Effects of PTX on thrombin- and TRag-evoked ERK1/2
phosphorylation and proliferation in rat astrocytes. Serum-starved
astrocytes were preincubated with PTX (200 ng/ml) for 24 h before
5 min of stimulation with thrombin (1 U/ml), TRag (1 µM), or EGF (50 ng/ml). A: representative blot of ERK1/2 phosphorylation.
B: quantification by densitometer. Data represent means ± SE of at least 3 experiments. **P < 0.01 vs. cells
exposed to thrombin or TRag alone. C: serum-starved
astrocytes were preincubated with PTX (200 ng/ml) for 24 h before
3 h of stimulation with thrombin (10 U/ml) or TRag (10 µM).
Proliferation is expressed as percent change compared with control.
Data represent means ± SE (n 6 wells/condition). *P < 0.05 vs. cells exposed to
thrombin or TRag alone.
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Association of InsP3/Ca2+
with thrombin-induced proliferation and ERK phosphorylation.
Activation of PAR-1 results in elevation of intracellular
Ca2+ in astrocytes through both Ca2+ release
from internal stores and Ca2+ influx (7, 58,
59). Measurements of intracellular Ca2+ in
astrocytes showed that the transient rise in
[Ca2+]i elicited by short-term stimulation
with thrombin and TRag can be nearly completely blocked by application
of U-73122 (5 µM), a PLC inhibitor, and 2-APB (500 µM), a
noncompetitive antagonist of the intracellular InsP3
receptor (see data in Table 1). Because activation of PLC and
liberation of InsP3 are events upstream of the
Ca2+ release from intracellular stores, our results suggest
that the initial Ca2+ response induced by short pulses of
thrombin and TRag is mainly caused by Ca2+ release. This
rise in [Ca2+]i may also be involved in the
subsequent mitogenic signaling cascade induced by PAR-1 activation.
To determine whether PLC and InsP3 are upstream factors of
the proliferative effect, we employed U-73122 and 2-APB in the ERK
phosphorylation and proliferation assay. As shown in Fig. 5, pretreatment with U-73122 (5 µM) for
10 min significantly attenuated thrombin- and TRag-induced ERK1/2
phosphorylation. Interestingly, proliferation in astrocytes was reduced
to a similar degree.

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Fig. 5.
Involvement of PLC in thrombin- and TRag-evoked ERK1/2
phosphorylation and proliferation in rat astrocytes. Serum-starved
astrocytes were preincubated with U-73122 (5 µM) or U-73343 (5 µM)
for 15 min before 5 min of stimulation with thrombin (1 U/ml), TRag (1 µM), or EGF (50 ng/ml). A: representative blot of ERK1/2
phosphorylation from 1 experiment. B: quantification by densitometer.
Data represent means ± SE of at least 3 experiments.
*P < 0.05 vs. cells exposed to thrombin or TRag alone.
C: serum-starved astrocytes were preincubated with U-73122
(5 µM) or U-73343 (5 µM) for 15 min before 3 h of stimulation
with thrombin (10 U/ml) or TRag (10 µM). Proliferation is expressed
as percent change compared with control. Data represent means ± SE (n 6 wells/condition). *P < 0.05 vs. cells exposed to thrombin or TRag alone.
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U-73343 (5 µM), an inactive analog of U-73122 that is frequently used
as a control compound for U-73122, did not affect thrombin- and
TRag-mediated [Ca2+]i increase, ERK1/2
phosphorylation, and proliferation in astrocytes (data in Table 1 and
Fig. 5). EGF-induced ERK1/2 activation was not influenced by U-73122
(Fig. 5, A and B). These results confirm the
conclusion that the attenuation seen with U-73122 is not a nonspecific inhibition.
Figure 6 shows that 2-APB (500 µM)
exerts much stronger inhibitory effects than U-73122 (Fig. 5). Both
thrombin- and TRag-induced ERK1/2 phosphorylation and enhancement of
proliferation were blocked substantially by treatment with 2-APB. A
small inhibition by 2-APB was observed in EGF-evoked ERK
phosphorylation, which is negligible when compared with the strong
inhibition of thrombin- and TRag-induced responses. In addition,
pretreatment with Ca2+ ionophore A-23187 (300 nM) was
tested to elucidate the role of Ca2+ in thrombin-induced
cell proliferation. A-23187 can induce significant ERK1/2
phosphorylation and proliferation in astrocytes, respectively (Fig. 6),
providing further evidence for the contribution of intracellular Ca2+. Taken together, these data demonstrate the
involvement of PLC and InsP3/Ca2+ in thrombin-
and TRag-induced mitogenic response in rat astrocytes.

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Fig. 6.
Involvement of inositol 1,4,5-trisphosphate
(InsP3)/Ca2+ in thrombin- and TRag-evoked
ERK1/2 phosphorylation and proliferation in rat astrocytes.
Serum-starved astrocytes were preincubated with
2-aminoethoxydiphenylborate (2-APB; 100 µM) for 15 min before 5 min
of stimulation with thrombin (1 U/ml), TRag (1 µM), or EGF (50 ng/ml); in addition, 5 min of exposure to A-23187 (300 nM) was tested.
A: representative blot of ERK1/2 phosphorylation from 1 experiment. B: ERK1/2 phosphorylation induced by thrombin or
TRag alone was considered as 100%. Data represent means ± SE of
at least 3 experiments. **P < 0.01 vs. cells exposed
to thrombin or TRag alone. C: serum-starved astrocytes were
preincubated with 2-APB (100 µM) for 15 min before 3 h of
stimulation with thrombin (10 U/ml) or TRag (10 µM), or only for
3 h with A-23187 (300 nM). Proliferation is expressed as percent
change compared with control. Data represent means ± SE (n
6 wells/condition). **P < 0.01 vs. cells
exposed to thrombin or TRag alone.
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Role of PI 3-kinase within thrombin-induced proliferation and ERK
phosphorylation.
In the present study we have shown that ERK1/2 phosphorylation in
response to thrombin and TRag in astrocytes involves PTX-sensitive G
proteins such as Go/Gi. Some reports suggested
that Gi proteins can activate MAP kinases through their
G
subunits, an effect that was mediated via PI 3-kinase (8,
31). Therefore, we tested the role of PI 3-kinase in thrombin-
and TRag-induced cellular events in astrocytes. As shown in Table 1,
treatment of astrocytes with the PI 3-kinase inhibitor wortmannin (5 µM) attenuated thrombin- and TRag-induced Ca2+ response
by 39 and 63%, respectively. Results in Fig.
7 demonstrate substantial inhibition by
wortmannin of thrombin- and TRag-induced ERK1/2 phosphorylation (by at
least 81 and 98%) and proliferation (by 81 and 83%), confirming the
involvement of PI 3-kinase. In this case, only a small inhibition was
observed with EGF-stimulated ERK1/2 phosphorylation by wortmannin.

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Fig. 7.
Involvement of phosphatidylinositol (PI) 3-kinase in
thrombin- and TRag-evoked ERK1/2 phosphorylation and proliferation in
rat astrocytes. Serum-starved astrocytes were preincubated with
wortmannin (5 µM) for 15 min before 5 min of stimulation with
thrombin (1 U/ml), TRag (1 µM), or EGF (50 ng/ml). A:
representative blot of ERK1/2 phosphorylation from 1 experiment.
B: quantification by densitometer. Data represent means ± SE of at least 3 experiments. **P < 0.01 vs. cells
exposed to thrombin or TRag alone. C: serum-starved
astrocytes were preincubated with wortmannin (5 µM) for 15 min before
3 h of stimulation with thrombin (10 U/ml) or TRag (10 µM).
Proliferation is expressed as percent change compared with control.
Data represent means ± SE (n 6 wells/condition).
**P < 0.01 vs. cells exposed to thrombin or TRag
alone.
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Involvement of PKC in thrombin-induced proliferation and ERK
phosphorylation.
Signaling from GPCR to the ERK/MAPK cascade can be transmitted by
several distinct pathways, some of which involve PKC (36, 50). PKC is activated by diacylglycerol, which is generated during the hydrolysis of phosphatidylinositol 4,5-bisphosphate after
PLC activation. To examine the role of PKC in thrombin- and
TRag-mediated effects in astrocytes, we pretreated cells with the PKC
inhibitor GF-109203X (1 µM). Cells treated with GF-109203X showed a
decreased Ca2+ response to thrombin and TRag stimulation,
with 43 and 55% reduction, respectively.
Furthermore, biochemical studies showed that thrombin- and TRag-induced
ERK1/2 phosphorylation and proliferation were significantly inhibited
by pretreatment with GF-109203X, as shown in Fig.
8. ERK1/2 phosphorylation was reduced by
73 and 74%, and proliferation by 59 and 74%. No inhibition was
observed with GF-109203X on EGF-stimulated ERK activation, indicating
that PKC specifically plays an important role in PAR activation-induced
responses in astrocytes.

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Fig. 8.
Involvement of PKC in thrombin- and TRag-evoked ERK1/2
phosphorylation and proliferation in rat astrocytes. Serum-starved
astrocytes were preincubated with GF-109203X (1 µM) for 15 min before
5 min of stimulation with thrombin (1 U/ml), TRag (1 µM), or EGF (50 ng/ml). A: representative blot of ERK1/2 phosphorylation
from 1 experiment. B: quantification by densitometry. Data
represent means ± SE of at least 3 experiments.
**P < 0.01 vs. cells exposed to thrombin or TRag
alone. C: serum-starved astrocytes were preincubated with
GF-109203X (1 µM) for 15 min before 3 h of stimulation with
thrombin (10 U/ml) or TRag (10 µM). Proliferation is expressed as
percent change compared with control. Data represent means ± SE
(n 6 wells/condition). *P < 0.05 vs.
cells exposed to thrombin or TRag alone.
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Question of transactivation of EGF receptor in thrombin-induced
proliferation and ERK phosphorylation.
Recently emerging evidence has indicated that in certain cell types,
the mitogenic effect of thrombin stimulation can be mediated through
transactivation of the EGF receptor (9, 27). To verify the
nature of the mitogenic actions of thrombin and PAR-1 in astrocytes, we
used AG1478 (5 µM), an inhibitor of EGF receptor kinase.
First, as shown in Fig. 9, A
and B, we found that stimulation of cells for 5 min
with EGF also induced the phosphorylation of ERK1/2. According to the
density of the phosphorylation signal on the blot, EGF (50 ng/ml;
~0.8 nM) caused a more robust ERK1/2 activation than thrombin (1 U/ml) and TRag (1 µM). The EGF effect can be totally suppressed by
pretreatment with AG1478 and the MEK inhibitor PD-98059. While inducing
a smaller maximum activation of ERK1/2 than EGF under the experimental
conditions, thrombin- and TRag-stimulated ERK1/2 activation was only
partially blocked by AG1478. This result, however, is not yet
sufficient evidence to clarify the question of whether or not EGF
receptor transactivation occurs following PAR-1 activation. Next,
AG1478 was tested in the proliferation assay. As shown in Fig.
9C, similar to the inhibition of ERK1/2 phosphorylation by
AG1478, EGF induced-proliferation was completely inhibited by
pretreatment of AG1478, whereas only a small (statistically
insignificant) reduction was observed with thrombin- and
TRag-stimulated proliferation. This result suggests the possibility
that AG1478 might exert some small, nonspecific inhibition of tyrosine
kinase phosphorylation in astrocytes.

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Fig. 9.
Effects of AG1478 on ERK1/2 phosphorylation and astrocyte
proliferation induced by EGF and PAR-1 activation. Serum-starved
astrocytes were preincubated without or with AG1478 (5 µM) for 15 min
before 5 min of stimulation with EGF (50 ng/ml), thrombin (1 U/ml), or
TRag (1 µM). A: representative blot of ERK1/2
phosphorylation from 1 experiment. B: ERK1/2 phosphorylation
induced by EGF, thrombin, or TRag alone was considered as 100%. Data
represent means ± SE from at least 3 experiments.
**P < 0.01 vs. cells exposed to EGF alone.
C: serum-starved astrocytes were preincubated without or
with AG1478 (5 µM) for 15 min before 3 h of stimulation with EGF
(50 ng/ml), thrombin (10 U/ml), or TRag (10 µM). Proliferation is
expressed as percent change compared with control. Data represent
means ± SE (n 6 wells/condition).
**P < 0.01 vs. cells exposed to EGF alone.
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Therefore, the concentration dependence of inhibition by AG1478 was
measured for EGF- and PAR-1-induced ERK1/2 phosphorylation. As shown in
Fig. 10, the potency of AG1478 to
inhibit PAR-1-mediated activation of ERK1/2 was almost an order of
magnitude greater than its potency to inhibit EGF-induced ERK1/2
activation. However, maximally, AG1478 caused an ~55% inhibition of
ERK activation, whereas this compound was able to inhibit
EGF-stimulated ERK phosphorylation completely. Given the known
specificity of AG1478 for the EGF receptor kinase, this result could be
interpreted to indicate that 55% of the ability of PAR-1 to activate
ERK1/2 could conceivably involve the EGF receptor, even though
autophosphorylation of the receptor cannot be detected (see below).
Alternatively, the ability of AG1478 to inhibit PAR-1-stimulated ERK
activation by 55% might be due to its inhibition of a kinase distinct
from the EGF receptor. Whatever this interesting kinase might be, it is
very sensitive to the inhibitor AG1478.

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Fig. 10.
Concentration-dependent inhibition by AG1478 of
thrombin-, TRag-, and EGF-evoked ERK1/2 phosphorylation in rat
astrocytes. Serum-starved astrocytes were preincubated without or with
AG1478 for 15 min before 5 min of stimulation with EGF (50 ng/ml),
thrombin (1 U/ml), or TRag (1 µM). The phosphorylation of ERK1/2 was
expressed as a percentage of the phosphorylation of ERK1/2 by 5 min of
EGF (50 ng/ml), thrombin (1 U/ml), or TRag (1 µM) stimulation. Data
represent means ± SE of at least 3 experiments.
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To clearly determine whether the partial inhibitory effects of AG1478
on thrombin- or TRag-induced response were due to the inhibition of EGF
receptor activation subsequent to PAR-1 activation, we then examined
the phosphorylation status of the EGF receptor. In these experiments
the EGF receptor was analyzed by immunoprecipitation analysis. As shown
in Fig. 11A, 5 min of
stimulation with EGF elicited pronounced tyrosine phosphorylation of
the EGF receptor, as shown by probing EGF receptor immunoprecipitates
on the blot with phosphotyrosine antibodies. This EGF receptor
phosphorylation was totally blocked by pretreatment of astrocytes with
AG1478 (5 µM). In contrast, no signal of phosphorylated EGF receptor
was observed with either thrombin or TRag stimulation, indicating that
EGF receptor has minimal, if any, involvement in PAR-1-induced
astrocytic proliferation.

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Fig. 11.
Western blots showing the effect of thrombin, TRag, or
EGF on tyrosine phosphorylation of the EGF receptor. A:
detection of EGF receptor phosphorylation by immunoprecipitation (IP)
in rat astrocytes. Serum-starved astrocytes were preincubated without
or with AG1478 (5 µM) for 15 min before 5 min of stimulation with EGF
(50 ng/ml), thrombin (1 U/ml), or TRag (1 µM). EGF receptor was
immunoprecipitated from cell lysates and analysed by immunoblotting
with either anti-phosphotyrosine ( PY) or anti-EGF receptor ( EGFR)
antibody (Ab). B: detection of EGFR phosphorylation (pEGFR)
by immunoblotting in rat astrocytes. Serum-starved astrocytes were
preincubated without or with AG1478 (5 µM) for 15 min before 5 min of
stimulation with EGF (50 ng/ml), thrombin (1 U/ml), or TRag (1 µM).
Cell lysate obtained after stimulation was mixed with solvent of
methanol and aceton (1:1) at a ratio of 1:4 volumes. After incubation
at 37°C for 15 min, the mixture was centrifuged at 13,000 rpm for 15 min. The pellet was dried and dissolved in SDS sample buffer. The
samples were heated for 5 min at 95°C before being loaded onto
polyacrylamide gels. pEGFR was analyzed by immunoblotting with
anti-pEGFR (tyrosine 1068) (upper blot). Lower blot
shows equal loading of protein by detection of EGFR. Experiments
in A and B were repeated 3 times with identical
results. C: correlation between the concentration-effect
curve of EGF-stimulated ERK phosphorylation and EGF-meditaed EGFR
tyrosine phosphorylation in rat astrocytes. Serum-starved astrocytes
were stimulated for 5 min with EGF. pERK1/2 were detected by Western
blot as described previously (upper blot). EGFR was
immunoprecipitated from cells lysates and analyzed by immunoblotting
with PY antibody (lower blot). Experiments were repeated
3 times with identical results.
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Moreover, to corroborate these results, we have also employed an
immunoblotting assay using an antibody that binds to phospho-EGF receptor specifically at tyrosine 1068. Tyrosine 1068 is one of the
major sites accounting for EGF receptor autophosphorylation (25). Phospho-tyrosine 1068 of activated EGF receptor is a
direct binding site for the Grb2/SH2 domain (49). This
binding results in Ras activation through a Grb2/Sos-1 signaling
mechanism (65). Phosphorylation of EGF receptor at
tyrosine 1068 has also been implicated under transactivation by GPCR
(60). However, in our study, as shown in Fig.
11B, no phosphorylation of the EGF receptor residue tyrosine
1068 was detected after either thrombin or TRag stimulation. Because
AG1478 failed to inhibit PAR-1-induced astrocyte proliferation, EGF
receptor transactivation did not appear to be involved in the mitogenic
action of PAR-1.
Finally, to exclude the possibility that a small degree of
tyrosine phosphorylation of EGF receptor that might be below the level
detectable by the Western blot would be sufficient for maximal MAPK
activation, the correlation between the concentration-effect curve of
EGF-stimulated ERK phosphorylation and EGF-mediated EGF receptor
tyrosine phosphorylation was examined. As shown in Fig. 11C,
1 ng/ml EGF elicited a detectable signal of phosphorylated EGF receptor
together with a level of ERK1/2 phosphorylation that was similar to
that induced by stimulation with thrombin (1 U/ml), whereas thrombin
stimulation yielded no EGF receptor phosphorylation. The ERK1/2
phosphorylation by 1 ng/ml EGF and by thrombin (1 U/ml) was 65 and 80%
of that induced by 50 ng/ml EGF, respectively. Taken together, these
results demonstrate that the mitogenic response induced by thrombin and
TRag is mediated primarily through the PAR-1-connected signaling
pathways, independent of EGF receptor transphosphorylation.
 |
DISCUSSION |
The goal of the present study is to further the understanding of
the signal transduction mechanisms underlying thrombin-induced proliferation in astrocytes. Previously, we have shown that rat astrocytes functionally coexpress all four subtypes of PARs. A comparable proliferation was obtained when astrocytes were exposed either for 3 or 24 h to thrombin, TRag, or PAR-2-AP
(62). This finding suggests that the initial signaling
induced by activation of PAR-1 or PAR-2, especially PAR-1, is
sufficient to trigger the proliferation of astrocytes. These results
are in line with previous reports that thrombin acts as a mitogen for
astrocytes through PAR-1 (18). In other cell types, such
as human cultured tracheal smooth muscle cells (36), mouse
lung fibroblasts (56), and airway smooth muscle cells
(44), it has been clearly shown that MAPKs are activated
by thrombin, leading to cell proliferation. Therefore, it was
hypothesized that ERK1/2 may also play a central role in the
thrombin/PAR-1-evoked proliferative effect in astrocytes.
It was shown in the present study that stimulation by thrombin
activated ERK1/2 in astrocytes. Interestingly, we found that the
respective PAR-AP acted in a mode similar to the protease to activate
ERK1/2, but to a different degree. TRag, a synthetic specific agonist
of PAR-1, induced a response resembling in amplitude that of thrombin,
not only with ERK1/2 activation (95% of the maximum response inducible
by thrombin) but also with intracellular Ca2+ mobilization
and astrocytic proliferation (62). However, PAR-2-AP exhibited a smaller potency on ERK1/2 phosphorylation and
proliferation. In accordance with our previous data showing that the
Ca2+ signal evoked by PAR-3 and PAR-4-AP was relatively
weak and that both peptides lack the ability to induce proliferation in
astrocytes (62), only a small, almost negligible response
was observed on ERK1/2 phosphorylation in this study (Fig.
2F). These results suggest that activation of PARs
stimulates ERK1/2 phosphorylation in astrocytes. Furthermore, the data
show a close correlation between the amplitude of the Ca2+
response and the extent of ERK1/2 activation as well as proliferation induced by activation of PARs in astrocytes. We have demonstrated that
thrombin utilizes PAR-1, -3, and -4 for signal transduction in
astrocytes (62), but it seems that PAR-1 is the most
prominent receptor among PARs for mediating the cellular consequence of thrombin stimulation in astrocytes.
Moreover, PD-98059, an inhibitor of the ERK activator MEK, was employed
to elucidate the relationship of ERK1/2 phosphorylation and
proliferation induced by thrombin and TRag. PD-98095 has been shown to
block ERK stimulation and to inhibit growth factor-induced proliferation in Swiss 3T3 mouse fibroblasts and rat kidney cells. PD-98095 is highly selective for MEK, as evidenced by its failure to
inhibit 18 other serine/threonine protein kinases in vitro and in vivo,
including the ERK homolog Jun NH2-terminal kinase (1). The result that PD-98059 abolished effects of both
thrombin and TRag in astrocytes supports our hypothesis that
proliferation induced by PAR-1 activation is mediated through ERK1/2
activation in astrocytes.
G protein-linked signaling from PAR-1 to ERK1/2 in astrocytes.
PAR-1 can couple to PTX-sensitive and -insensitive G proteins
(19). There is considerable evidence that PTX-sensitive G proteins mediate mitogenic responses and activation of MAPK cascades elicited by a variety of G protein-coupled receptors (24,
61). In cultured rat astrocytes, we found that pretreatment of
cells with PTX, which inhibits Go/Gi proteins,
partially attenuated all responses induced by thrombin and TRag: the
Ca2+ signal, ERK1/2 activation, and proliferation. A
similar effect of PTX on thrombin and thrombin receptor-activating
peptide (TRAP-14)-induced DNA synthesis has been reported in astrocytes
(10). The specificity of PTX and the other inhibitors
discussed below was proven in the present study by positive controls
for activation of ERK1/2. In addition, results presented in Table 1
also showed positive evidence for the inhibitory activities of PTX,
U-73122, and 2-APB on Ca2+ response induced by PAR-1 activation.
The mechanism of receptor tyrosine kinase (RTK)-stimulated mitogenic
signaling involves the formation of complexes between the guanine
nucleotide exchange protein Sos and the adaptor protein Grb2 with
another tyrosine-phosphorylated adaptor protein, Shc. Recent studies
have shown that some GPCRs utilize the same effectors as the RTK
pathway (e.g., Shc-Grb-Sos), resulting in Ras and MAPK activation. This
cascade is initiated by 
-subunits and involves a
wortmannin-sensitive PI 3-kinase (22, 43).
Gi-coupled receptors have been proposed to regulate
Ras-dependent signaling cascades through the release of G protein

-subunits. We tried to clarify the issue of whether G
and/or PI 3-kinase are involved in thrombin- and TRag-stimulated
Ca2+ signal, ERK1/2 phosphorylation, and proliferation.
Therefore, astrocytes were pretreated with wortmannin, the inhibitor of
PI 3-kinase. Our results (Fig. 7) showed that the rise in
[Ca2+]i was partially suppressed by
wortmannin, whereas ERK1/2 phosphorylation and proliferation were
strongly blocked. Obviously, PI 3-kinase plays a decisive role in the
signaling pathways initiated by thrombin and TRag stimulation in
astrocytes. Furthermore, these results provide indirect proof for the
possible role of PTX-sensitive G protein 
-subunits in astrocytes.
Such PI 3-kinase-mediated thrombin-induced cell proliferation was also
observed in human airway smooth muscle cells (32), aortic
smooth muscle cells (51), and human tracheal smooth muscle
cells (36). The finding that both RTK and GPCR pathways
can activate a similar set of signal transducers indicates more
parallels than originally thought.
However, the fact that inhibition by PTX of the increase in
[Ca2+]i and ERK1/2 phosphorylation was only
partial in astrocytes indicates the participation also of
PTX-insensitive G proteins. In fibroblasts, thrombin initiates the
mitogenic signaling pathway by coupling to both PTX-sensitive and
-insensitive G proteins (33). PTX-insensitive G proteins
like Gq,11 give rise to the activation of PLC
and the
generation of InsP3 and diacylglycerol, which in turn lead to the mobilization of intracellular Ca2+ and activation of
PKC. The data presented in this study have shown that the PLC inhibitor
U-73122 substantially suppressed the Ca2+ mobilization and,
to a lesser degree, prevented the ERK1/2 phosphorylation and
proliferation induced by thrombin and TRag. These downregulation effects were due to the specific inhibition of PLC because U-73343, the
inactive analog of U-73122, was ineffective. Meanwhile, the InsP3 receptor antagonist 2-APB, which has been shown to
inhibit InsP3 receptor-induced
[Ca2+]i elevation in a variety of cell types
(37, 55), exerted potent inhibitory effects on thrombin-
and TRag-stimulated cellular responses as well. Moreover, significant
ERK phosphorylation and cell proliferation were also observed with the
stimulation by the Ca2+ ionophore A-23187. These results
further demonstrate that PLC and InsP3/Ca2+ act
as upstream factors of ERK1/2 phosphorylation in astrocytes, which is
in line with ERK1/2 activation induced by endothelin or glutamate in
astrocytes (50, 52). In fact, Ca2+ signaling
has been implicated as an important growth signal in many cell types
(3, 40). Several Ca2+-dependent kinases like
proline-rich tyrosine kinase 2 (Pyk2) and
Ca2+/calmodulin-dependent protein kinase II (CaMKII) have
also been demonstrated to be involved in the MAPK activation pathway in some cell types (41, 48). Whether these
Ca2+-regulated kinases act as mediators in the PAR-1
signaling cascade as well is currently under investigation.
A number of studies with various GPCRs have demonstrated two signaling
pathways from the receptor to the activation of MAPK: a PTX-sensitive,
Ras-dependent pathway mediated by G
and, in addition, a
PTX-insensitive, Ras-independent pathway regulated by PKC (11,
23, 29). In the present study, preincubation of astrocytes with
PKC inhibitor GF-109203X significantly attenuated the thrombin- and
TRag-induced increase in [Ca2+]i, ERK1/2
phosphorylation, and proliferation, implying the essential role of PKC
for astrocytic proliferation. In fact, this implication of PKC
involvement has been further supported by the inhibitory effects of the
PLC inhibitor U-73122 and the InsP3 receptor antagonist 2-APB, because they are well-established PKC activators. Our previous results have also shown that activation of PKC is required to maintain
the refilling of intracellular Ca2+ stores for sustained
thrombin-induced [Ca2+]i oscillations in rat
glioma cells, because addition of GF-109203X irreversibly suppressed
thrombin-induced [Ca2+]i oscillations
(57). The inhibitory effects of GF-109203X obtained in
this study additionally support the notion that
[Ca2+]i is involved in the ERK1/2
phosphorylation and proliferation induced by thrombin and TRag in
astrocytes as well.
Possible involvement of EGF receptor transactivation in
PAR-1-ERK1/2 activation in astrocytes.
Despite the fact that activation of GPCRs is able to stimulate
mitogenesis in a variety of cell types, several groups have recently
implicated the EGF receptor as a necessary signaling component in
response to GPCR activation. An alternative mechanism proposed by Daub
et al. (9) suggested that GPCRs activate MAPK in Rat-1
fibroblasts through transactivation of the EGF receptor. They further
proved that EGF receptor transactivation upon GPCR stimulation involves
heparin-binding EGF-like growth factor and a metalloprotease activity
that is rapidly induced upon GPCR-ligand interaction (46).
So far, thrombin has been found to cause EGF receptor transactivation
in diverse cell types such as HaCaT keratinocytes, COS-7 cells, mouse
astrocytes, and rat smooth muscle cells (8, 27).
We speculated that the transactivation mechanism may also account for
the PAR activation-induced cellular consequences in rat astrocytes,
because initially we found that EGF receptor kinase inhibitor AG1478
partially blocked ERK1/2 phosphorylation induced by thrombin and TRag.
However, lack of significant inhibition of thrombin- and TRag-induced
proliferation with AG1478 treatment raised the alternative possibility
that thrombin and TRag have their own signaling pathways distinct from
EGF receptor transactivation. Our comprehensive and detailed
experiments trying to detect the phosphorylated EGF receptor further
demonstrated that EGF, but not thrombin and TRag, stimulated EGF
receptor phosphorylation in rat astrocytes. Interestingly, Crouch et
al. (6) very recently showed in Swiss 3T3 cells that
thrombin has no direct effect on the activation state of the EGF
receptor or of its downstream effectors, although thrombin causes
clustering and sensitization of EGF receptor in migrating cells. They
showed that DNA synthesis induced by thrombin was resistant to
inhibition by AG1478, being only partially inhibited. Similarly, a
partial blockade of thrombin-induced ERK1/2 phosphorylation by AG1478
was also observed in their study. AG1478 inhibits the kinase function
of EGF receptor by interacting with the ATP binding site in the
1-10 nM range, but its exact mode of inhibition corresponding to
the protein substrate is yet unknown (17, 35, 42).
Therefore, the partial suppression of thrombin and TRag responses by
AG1478 in astrocytes might also be attributed to the inhibition of some
unknown kinases. Similarly, in COS-7 cells, PI 3-kinase was confirmed
to function as an upstream effector of Ras in GPCR-mediated MAPK
stimulation, whereas PI 3-kinase was not involved in cross talk between
GPCRs and the EGF receptor. However, an increase in PI 3-kinase
activity associated with Grb2 upon LPA treatment was also reversed by
AG1478 pretreatment (8).
Metalloproteinases have been proposed as a key intermediate for the
release of heparin-binding EGF leading to transactivation of EGF
receptor (9, 27). Therefore, we considered this
possible involvement and made some experiments with maximastat, an
inhibitor of metalloproteinase 9. We did not see any inhibition of
thrombin- and TRag-induced ERK phosphorylation by this inhibitor (data
not shown). This result supports our interpretation that no EGF
receptor phosphorylation could be induced by PAR-1 activation. We still do not know whether EGF receptor transphosphorylation on another tyrosine residue, e.g., Y845, might be involved in the ability of PAR-1
to activate ERK1/2. Although increasing evidence has indicated the
cross talk between EGF receptor and GPCRs, transactivation of RTKs does
not seem to be a general prerequisite for the activation of MAPK by
GPCRs in all cell types, which is evidenced in rat aortic myocytes by
5-hydroxytryptamine stimulation (2), in human epidermoid
carcinoma cells by bradykinin stimulation (20), in smooth
muscle cells by histamine H1 receptor activation
(47), and in human embryonic kidney cells by opioid
receptor activation (30).
In summary, in this report we have elucidated the mechanism by which
thrombin and TRag induce astrocytic proliferation. The pathways
established are summarized in the scheme shown in Fig. 12. We have demonstrated that thrombin
and TRag induce a mitogenic stimulus via ERK1/2 activation only through
G protein-linked signaling, i.e., the PTX-sensitive G protein (
subunits)-PI 3-kinase branch and the
Gq-PLC-(InsP3 receptor) Ca2+-PKC
pathway but deliver very little or most likely no signal to EGF
receptor tyrosine kinase to evoke their mitogenic response. Our results
suggest that transactivation of EGFR might contribute only in some cell
types to GPCR-mediated mitogenic signaling.

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Fig. 12.
Proposed intracellular transduction mechanisms underlying
thrombin/PAR-1-induced activation of ERK1/2 and proliferation in
astrocytes. Activation of the PAR-1 (7 transmembrane-spanning
domains) by thrombin or the selective PAR-1-AP TRag results in
both PTX-sensitive and PTX-insensitive G proteins mediating the
activation of ERK1/2 cascades leading to proliferation. On one hand,
thrombin can activate ERK1/2 via PTX-sensitive G proteins and
downstream activation of a tyrosine kinase-dependent process, probably
through Ras- and Raf-dependent steps. On the other hand, thrombin
activates ERK1/2 via PTX-insensitive G proteins by activation of PLC,
resulting in intracellular Ca2+ mobilization and PKC
activation, probably through some Ca2+-dependent kinases
like Pyk2 leading to subsequent ERK1/2 phosphorylation. PAR-1 and EGF
receptor may recruit some common signaling complex leading to Ras
activation. However, the EGFR does not seem to participate in PAR-1
signaling in rat astrocytes. DAG, diacyl glycerol; IP3,
inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol
4,5-bisphosphate.
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ACKNOWLEDGEMENTS |
We thank Stephanie Balcaitis for help with the language of the manuscript.
 |
FOOTNOTES |
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Graduiertenkolleg für "Biologische
Grundlagen von Erkrankungen des Nervensystems"), Land Sachsen-Anhalt
(2923A/0028H), Bundesministerium für Bildung und Forschung (01-ZZ
9505), and Fonds der chemischen Industrie.
Present address of J. J. Ubl: Ludwig-Maximilians Universität
München, Institut für Neuropathologie, 81377 München, Germany.
Address for reprint requests and other correspondence: G. Reiser, Otto-von-Guericke-Universität Magdeburg, Medizinische
Fakultät, Institut für Neurobiochemie, Leipziger Str. 44, 39120 Magdeburg, Germany (E-mail:
georg.reiser{at}medizin.uni-magdeburg.de).
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.
June 20, 2002;10.1152/ajpcell.00001.2002
Received 2 January 2002; accepted in final form 18 June 2002.
 |
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