(Received for publication, May 30, 1995; and in revised form, June 26, 1995)
From the
Thrombin stimulation of 1321N1 astrocytoma cells leads to
Ras-dependent AP-1-mediated transcriptional activation and to DNA
replication. In contrast to what has been observed in most cell
systems, in 1321N1 cells these responses are pertussis
toxin-insensitive. The pertussis toxin-insensitive G-protein G has been implicated in cell growth and transformation in
different cell systems. We have examined the potential role of this
protein in AP-1-mediated transcriptional activation and DNA synthesis
in 1321N1 cells. Transient expression of an activated
(GTPase-deficient) mutant of G
increased
AP-1-dependent gene expression. This response was inhibited by
co-expression of a dominant negative Ala-15 Ras protein. To determine
whether the pertussis toxin-insensitive G
protein is
involved in the thrombin-stimulated DNA synthesis, an inhibitory
antibody against the C-terminal sequence of G
subunit
was microinjected into 1321N1 cells. Microinjection of the
anti-G
resulted in a concentration-dependent
inhibition of thrombin-stimulated DNA synthesis. In contrast,
microinjection of nonimmune IgG or an antibody directed against the C
terminus of G
did not reduce the mitogenic response to
thrombin. Furthermore, microinjection of the anti-G
antibody had no effect on fibroblast growth factor-stimulated DNA
synthesis. These results demonstrate a specific role for G
in the mitogenic response to thrombin in human astroglial cells.
Thrombin is a potent mitogen for fibroblasts, astrocytes, and other cell lines (Van Obberghen-Schilling et al., 1985; Cavanaugh et al., 1990; Hung et al., 1992; LaMorte et al., 1993a, 1993b). Thrombin cleaves and activates a seven-transmembrane-spanning receptor to trigger G-protein-mediated stimulation of downstream effectors (Vu et al., 1991). The actions of thrombin in 1321N1 human astrocytoma cells have been well characterized. In these cells, thrombin stimulates phospholipase C activity leading to mobilization of intracellular calcium, diglyceride generation, and redistribution of protein kinase C (Jones et al., 1989; Nieto et al., 1994). Thrombin receptor activation also leads to a biphasic increase in c-jun mRNA, an associated increase in AP-1 DNA binding activity, and a marked increase in AP-1-mediated gene expression and DNA synthesis (Trejo et al., 1992; LaMorte et al., 1993b). Ras function is required for the mitogenic effect of thrombin as well as thrombin-induced AP-1 transcriptional activity (LaMorte et al., 1993b).
There is substantial information about the
signaling pathways downstream of thrombin receptor activation; however,
it is not known which of the heterotrimeric G-proteins couples thrombin
to the mitogenic pathway. In fibroblasts, thrombin-stimulated DNA
synthesis is sensitive to pertussis toxin (PTX) ()(Chambard et al., 1987; van Corven et al., 1993). Experiments
using microinjected antibodies against G
or
G
suggested that the mitogenic effect of thrombin in
CCL39 and in 3T3 fibroblasts is mediated through these G-proteins
(LaMorte et al., 1993a; Baffy et al., 1994). However,
in 1321N1 astrocytoma cells, thrombin-stimulated DNA synthesis and
thrombininduced gene expression are not inhibited by pertussis toxin
treatment, (
)suggesting that G-proteins of the
G
/G
family do not mediate these responses. The
PTX-insensitive G-proteins G
and/or G
have
also been reported to function in mitogenic signaling by thrombin and
bradykinin in fibroblasts (LaMorte et al., 1993a; Baffy et
al., 1994). However, studies comparing muscarinic and thrombin
receptor signaling mechanisms in astrocytes and CCL39 fibroblasts
demonstrate that, although both receptors interact with
G
/G
to stimulate phospholipase C, release
calcium, and activate protein kinase C, only thrombin can activate Ras
and elicit cell proliferation (Trejo et al., 1992; Seuwen et al., 1990).
These data suggest that activation
of G
and phospholipase C is insufficient to account for the
full mitogenic effects of thrombin and that other signaling pathways
are involved.
The G and G
subunits constitute a family of G-proteins distantly related to
the other G-protein
subunits (Strathmann and Simon, 1991). Both
G
and G
lack the cysteine residue
that renders these proteins susceptible to ADP-ribosylation by PTX. The
signaling pathways regulated by these G-proteins have not been
identified. Activation of these G-proteins has nonetheless been linked
to cell growth in several cell systems. Overexpression of G
in NIH3T3 cells leads to neoplastic transformation (Chan et
al., 1993), and expression of a GTPase-deficient mutant form of
G
and G
can efficiently transform
NIH3T3 cells (Jiang et al., 1993; Xu et al., 1993)
and Rat-1 fibroblasts (Voyno-Yasenetskaya et al., 1994a). In
addition, epidermal growth factor-stimulated mitogen-activated protein
kinase activity is enhanced in fibroblasts that express the activated
mutants of G
and G
(Voyno-Yasenetskaya et al., 1994b). Therefore, these
G-proteins appear to be good candidates to be involved in
PTX-insensitive signaling pathways leading to cell growth.
In the
present study we show that the activated mutant form of G induces AP-1-mediated transcriptional activation when transfected
in 1321N1 cells, and this effect requires Ras function. Furthermore, we
show that microinjection of an antibody against G
can
specifically block the mitogenic effect of thrombin. Taken together,
these results suggest that G
functions as a
PTX-insensitive mediator of thrombin-induced DNA synthesis in 1321N1
cells.
The thrombin receptor is a member of the G-protein-coupled
receptor superfamily and has been shown to couple to G,
G
, and G
proteins in different signaling
pathways (Vu et al., 1991; LaMorte et al., 1993a;
Baffy et al., 1994). In addition, the thrombin receptor has
been shown to couple to G
in platelets (Offermanns et
al., 1994). In 1321N1 cells thrombin stimulates the DNA binding
activity of the transcription complex AP-1 and results in an associated
increase in AP-1-mediated gene expression (Trejo et al.,
1992). This response cannot be explained by activation of either
G
or G
alone.
To test the possible
involvement of G
in AP-1-mediated gene expression we
examine the ability of an activated GTPase-deficient form of
G
to transactivate an AP-1-responsive reporter
plasmid (2
TRE-LUC). 1321N1 cells were transiently
co-transfected with plasmids containing the constitutively activated
(GTPase-deficient) mutant forms of G
(Q229L),
G
(R183C), or G
(R179C) or with a
control plasmid along with the 2
TRE-LUC reporter. The
activated G
caused a 6-7-fold increase in
luciferase activity relative to control transfected cells (Fig. 1A). Only a modest (<2-fold) stimulation was
seen with the activated G
and G
mutants. The
marked stimulatory effect of the activated G
mutant
suggests that G
protein may couple thrombin-receptor
activation to the induction of the expression of AP-1-responsive target
genes.
Figure 1:
Effect of G on 2
AP-1-luciferase gene expression. Subconfluent 1321N1
cells were transiently transfected as described under
``Experimental Procedures.'' A, cells were
transfected with 3 or 9 µg of either pCISG
12Q229L,
pCISG
qR183C, or pCISG
iR179C or backbone vector and assayed
for luciferase activity. B, cells were transfected with either
pCISG
12Q229L or pCISG
qR183C in the presence of Ala-15 Ras or
its backbone vector pZIP and harvested 48 h later. The cells were
subsequently assayed for luciferase activity. The -fold induction is
calculated by comparison with cells transfected with the control
plasmid and normalized to total protein. Each bar represents
the mean ± S.E. of data from three separate experiments, each
containing three to four replicates.
To examine the involvement of Ras in the stimulation of AP-1
gene expression, the Ala-15 dominant inhibitory mutant of Ras (Powers et al., 1989) was coexpressed with the activated forms of
either G or G
and the AP-1-sensitive
luciferase reporter plasmid (Fig. 1B). The
G
-induced increase in AP-1 transcriptional activity
was not affected by Ala-15 Ras. However, expression of the Ala-15 Ras
mutant led to a 70% reduction in the G
-induced
luciferase activity. These results demonstrate that G
stimulation of AP-1-dependent gene expression at least partially
requires Ras. Together with our previous observation that Ras is
required for thrombin stimulation of AP-1 activity and mitogenesis
(LaMorte et al., 1993b), these results suggest that
G
may couple the thrombin receptor to Ras activation.
To more specifically examine the involvement of G in thrombin-stimulated mitogenesis, we microinjected inhibitory
G
antibodies into intact 1321N1 astrocytoma cells.
The specificity of the antiserum was first verified by Western blot
using extracts of COS-7 cells transiently transfected with plasmids
encoding G
, G
, G
,
and LacZ (Fig. 2). The antiserum raised against a peptide
corresponding to the 11 C-terminal amino acids of G
(referred to as CT95 or anti-G
antibody)
recognized a protein of the expected size in COS-7 cells expressing
G
but did not detect other G-protein
subunits.
The CT95 antiserum also recognized a protein of the expected size in
1321N1 astrocytoma cells.
Figure 2:
Identification of G in
1321N1 astrocytoma cells by Western analysis. Extracts of COS-7 cells
transiently transfected with pCISG
12, pCISG
13, pCISG
q,
and control plasmid (pCISLacZ) and 1321N1 membrane extracts were
analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted
with CT95 antibody.
Serum-deprived 1321N1 cells
were microinjected with either the anti-G antibody,
nonimmune IgG, or anti-G
antibody (CT112). Three hours
later, the cells were treated with 0.5 unit/ml thrombin. The nucleotide
analog BrdUrd was added to the medium, and 24 h later the cells were
fixed. Injected cells were simultaneously assessed for the presence of
injected antiserum and for the nuclear incorporation of BrdUrd by
immunofluorescence microscopy (Fig. 3). Thrombin stimulated DNA
synthesis in 50 ± 3% of the cells while only 10 ± 2% of
unstimulated cells synthesized DNA (Table 1). Injection of the
CT95 antibody (10 mg/ml) reduced the proportion of cells in which
thrombin stimulated DNA synthesis to 23 ± 3% (Table 1).
There was a direct correlation between the amount of antibody injected
and the degree of inhibition of BrdUrd incorporation (Fig. 4).
Microinjection of 15 mg/ml CT95 antibody resulted in a complete
abolition of thrombin-stimulated DNA synthesis but had no effect on
basal BrdUrd incorporation in unstimulated cells. Preincubation of the
CT95 antibody with the peptide immunogen resulted in loss of the
ability of the anti-G
antibody to block
thrombin-induced DNA synthesis (data not shown). There was no
inhibition of the response to thrombin in cells injected with
equivalent concentrations of nonimmune IgG. Furthermore, microinjection
of an anti-G
did not reduce the number of cells
undergoing DNA synthesis in response to thrombin (Table 1). These
results clearly demonstrate that inhibitory antibodies directed against
G
can specifically block thrombin-induced DNA
synthesis in 1321N1 cells.
Figure 3:
Microinjection of CT95 antibody against
G in thrombin-stimulated 1321N1 cells. Cells were
plated on glass coverslips and starved 24 h prior to injection. Random
areas of quiescent cells were injected with control IgG (10 mg/ml) or
CT95 antibody (10 mg/ml). Cells were stimulated with 0.5 unit/ml
thrombin in DMEM. Representative examples of injected cells are shown
in panelsa (IgG) and b (CT95). Panels
c, e and d, f represent the same field as a and b, respectively. a, b, phase
contrast micrograph; c, d, fluorescent
photomicrographs depicting injected cells; e, f,
fluorescent photomicrographs depicting injected cells stained for
BrdUrd incorporation. Arrows indicate the injected
cells.
Figure 4: Percent of DNA synthesis in thrombin-stimulated 1321N1 cells injected with CT95 antibody. Cells were plated on glass coverslips and starved 24 h prior to injection. Quiescent cells were injected in random fields with CT95 and IgG at the concentrations indicated in the lowerpanel. Cells were stimulated with 0.5 unit/ml thrombin in DMEM. DNA synthesis was statistically analyzed using the standard error of proportion comparing injected cells and uninjected cells from the same coverslips. Fields of 200-1200 cells were counted for each point. The results presented represent the means of at least two experiments. Errorbars represent the 95% confidence interval calculated by using the standard error of proportion.
To further demonstrate the specificity of
the blocking effect of the CT95 antibody we examined the effect of this
antibody on induction of DNA synthesis by bFGF. The actions of bFGF are
presumed to be mediated through tyrosine phosphorylation and do not
utilize G-protein signaling pathways. Quiescent 1321N1 cells were
injected with either the anti-G antibody or control
nonimmune IgG and subsequently stimulated with 20 ng/ml bFGF or with
0.5 unit/ml thrombin. bFGF induced DNA synthesis in 22 ± 4% of
the cells (Table 2). Microinjection of the anti-G
antibody (10 mg/ml) did not inhibit the bFGF-stimulated DNA
synthesis. These results indicate that bFGF does not utilize
G
for mitogenic signaling and that the anti-G
antibody does not exert a generalized inhibitory effect upon DNA
synthesis. These data further strengthen the argument for the
specificity of action of the anti-G
antibody on the
response to thrombin.
We additionally examined the effect of the
CT95 antibody on the serum stimulation of DNA synthesis. The addition
of 5% fetal calf serum to serum-deprived 1321N1 cells stimulated DNA
synthesis in 76-90% of the cells (Table 2). Microinjection
of the anti-G antibody reduced the response to serum
by 45%. A similar effect was likewise observed by LaMorte et
al.(1992) with an anti-G
antibody in
serum-stimulated fibroblasts where the thrombin response is
PTX-sensitive. The inhibitory effect of the anti-G
antibody suggests that serum-induced DNA synthesis in 1321N1
cells is mediated in part through receptor(s) that function via
G
. This observation argues that G
is a
necessary cellular transducer for certain types of mitogens in addition
to thrombin.
Our previous work demonstrated that the
mitogenic response to thrombin in 1321N1 cells was pertussis
toxin-insensitive, and we suggested that an additional pathway besides
the G
/phospholipase C pathway was required. The results
presented here suggest that G
is the other transducer
mediating the mitogenic effect of thrombin in the 1321N1 cells. The
inhibition of thrombin-stimulated DNA synthesis by the G
antibody coupled with the activation by G
of
AP-1-dependent transcription reported here provides the first evidence
of a signaling pathway in which G
links a G-protein
receptor to cell growth. It also suggests a new linkage between a
G-protein pathway and the oncogene ras.
The mechanism by
which G affects cell growth is still not clear. Recent
data demonstrate that
released from stimulation of
G
-coupled receptors can mediate the activation of MAP
kinases acting on a Ras-dependent pathway (Winitz et al.,
1993; Albas et al., 1993; Faure et al., 1994; Crespo et al., 1994; Koch et al., 1994).
released
from G
by thrombin activation could mediate the mitogenic
response. However, the experiments reported here demonstrate that the
subunit of G
alone is able to induce AP-1-mediated
gene expression. Other studies show that G
or the
constitutively activated G
subunits are able to cause
cell transformation (Chan et al., 1993; Jiang et al.,
1993). It seems likely therefore that there is a pathway activated by
the
subunit of G
, which can lead to cell growth in
some systems.
The direct effector of G is not
known. Responses shown to be indirectly mediated via G
are
the activation of phospholipase A
(Xu et al.,
1993) and of a Na
-H
exchanger
(Dhanasekaran et al., 1994), which is also regulated by
G
(Voyno-Yasenetskaya et al., 1994c). Since the
activation of the Na
-H
exchanger
involves protein kinase C (Dhanasekaran et al., 1994) it is
possible that the activation of a particular isoform of protein kinase
C by the G
pathway may contribute to the mitogenic effect.
In addition, G
-mediated gene expression is at least
partially Ras-dependent, and thus G
may regulate
proteins responsible for Ras activation. Further work will be necessary
to clarify the G
effector pathway and the steps leading to
mitogenesis.