(Received for publication, August 31, 1995; and in revised form, October 5, 1995)
From the
Recent evidence suggests that reactive oxygen species (ROS) may function as second messengers in intracellular signal transduction pathways. We explored the possibility that ROS were involved in lysophosphatidic acid (LPA)-induced mitogen-activated protein (MAP) kinase signaling pathway in HeLa cells. Antioxidant N-acetylcysteine inhibited the LPA-stimulated MAP kinase kinase activity. Direct exposure of HeLa cells to hydrogen peroxide resulted in a concentration- and time-dependent activation of MAP kinase kinase. Inhibition of catalase with aminotriazole enhanced the effect of LPA on induction of MAP kinase kinase. Further, LPA stimulated ROS production in HeLa cells. These findings suggest that ROS participate in the LPA-elicited MAP kinase signaling pathway.
Reactive oxygen species (ROS), ()such as superoxide
(O
), hydrogen peroxide
(H
O
), and hydroxyl radical
(OH
), are potent microbicidal agents, but excess ROS
can also cause oxidative damage to macromolecules of host
cell(1) . Previous studies have shown that elevated levels of
ROS could trigger intracellular signaling transduction pathways that
may mediate cellular protective
responses(2, 3, 4) . In addition to their
roles in inflammatory and pathological processes, increasing evidence
suggests that ROS may function as second messengers in cytokine
(interleukin-1 and tumor necrosis factor
) and some growth factor
signal transduction pathways that regulate transcription factors such
as NF-
B and AP-1 (5, 6) .
Lysophosphatidic
acid (LPA) is released by activated platelets and is thought to be
responsible for much of the activity in serum that promotes cell growth
and adhesion(7, 8) . LPA elicits its biological
responses through a putative receptor that is coupled to heterotrimeric
G-proteins(9) . Several proximal signaling events are known to
be evoked by LPA, including phosphoinositide hydrolysis and
Ca mobilization, release of arachidonic acid,
inhibition of adenylate cyclase, and induction of protein tyrosine
phosphorylation(10, 11) . It is likely that some of
these signaling events cross-interact to induce synergistic responses.
LPA rapidly activates the mitogen-activated protein (MAP) kinase
pathway(11, 12, 13, 14) . MAP
kinases are serine/threonine-protein kinases regulated by dual tyrosine
and threonine phosphorylation. Three subfamilies of MAP kinases, MAPK,
JNK, and HOG, have been cloned (for reviews, see (15, 16, 17) ). The 42-kDa MAP kinase
(p42) also called extracellular
signal-regulated kinase 2 (ERK2)) and 44-kDa MAP kinase
(p44
, ERK1) are phosphorylated and activated by
highly specific MAP kinase kinase 1 and MAP kinase kinase 2 (MKK1/2) (18) . For simplicity, p42
and
p44
will be referred to as MAP kinase in this
report. MAP kinase has been shown to play a pivotal role in cell
proliferation and differentiation(19) . It has been shown that
the LPA-induced MAP kinase activation is sensitive to pertussis toxin
inhibition, indicating a critical role of a pertussis toxin-sensitive
G
-protein(11, 12, 14) . However,
these data did not exclude the contribution of other signaling events
to the LPA-induced MAP kinase activation. LPA triggers a biphasic
arachidonic acid release in HeLa cells. (
)The first phase of
the LPA-induced arachidonic acid release precedes the activation of MAP
kinase kinase.
Arachidonic acid is known to give rise to
ROS through its subsequent metabolism (20) and activation of
NADPH oxidase(21) , prompting us to investigate the possible
involvement of ROS in the LPA-stimulated MAP kinase activation pathway.
We present evidence here that demonstrates the involvement of ROS in
the LPA-induced MAP kinase signaling pathway.
In all cell types examined, p42 is
specifically phosphorylated and activated by dual specificity MKK1 and
MKK2 (MKK1/2). Total activity of MKK1/2 in the cells represents a valid
measurement for the activation state of the MAP kinase pathway in the
rapid activation phase(14, 23) . As illustrated in Fig. 1, LPA and EGF markedly stimulated the MKK1/2 activity in
HeLa cells. To test for the possible involvement of ROS in MKK1/2
activation induced by LPA, we examined the effect of antioxidant N-acetylcysteine on MKK1/2 activation. N-Acetylcysteine directly scavenges ROS and also increases the
intracellular levels of reduced glutathione (GSH). GSH is a hydroxyl
radical scavenger and a substrate of glutathione peroxidase which
degrades H
O
. N-Acetylcysteine has been
used extensively to study the role of ROS in signaling
pathways(6, 25, 26, 27) . An
inhibition by N-acetylcysteine can be taken as an indication
of the involvement of ROS. The LPA-stimulated MKK1/2 activity was
inhibited by 82 ± 4% (average of two experiments ± range)
in cells pretreated with N-acetylcysteine (30 mM),
suggesting that ROS are involved in the LPA-induced MKK1/2 activation (Fig. 1). A similar result was obtained in Rat-1 cells. A
lesser, but statistically significant, attenuation (38 ± 8% in
two experiments) by N-acetylcysteine of the EGF-stimulated
MKK1/2 activity was also observed (Fig. 1), but was not
investigated further in the current study.
Figure 1:
Inhibition by N-acetylcysteine
on MKK1/2 activation. Serum-starved HeLa cells were treated with or
without N-acetylcysteine (NAC, 30 mM) for 90
min, and left unstimulated(-) or stimulated with lysophosphatidic
acid (20 µM) or EGF (25 ng/ml) for 5 min. MKK1/2 activity
was determined using a kinase-defective p42 (K52R) as substrate. The kinase reaction was carried out at
30 °C for 15 min. Arrow, K52R
band.
To verify that the inhibitory effect of N-acetylcysteine is attributable to its ability to scavenge ROS, we examined the effects of two other ROS scavengers, dimethyl sulfoxide and ascorbic acid, on the LPA-stimulated MKK1/2 activity. Dimethyl sulfoxide is an effective hydroxyl radical scavenger(28) . Ascorbic acid blocks free radical chain reaction, but may also directly remove hydroxyl radical(29) . HeLa cells were pretreated with ascorbic acid (100 µM, 60 min) or dimethyl sulfoxide (4%, 20 min) and stimulated with LPA (10 µM, 5 min). The LPA-stimulated MKK1/2 activity was inhibited by 88 ± 7% by dimethyl sulfoxide and 38 ± 1% by ascorbic acid.
If ROS are the signaling molecules that mediate the
LPA-induced MKK1/2 activation, then an increase in intracellular
concentrations of ROS would be expected to mimic the effect of LPA on
MKK1/2 activation. HO
is the product of
superoxide dismutases and several oxidases in the cells. Thus, cells
that produce superoxide would also generate H
O
.
In contrast to superoxide, H
O
can diffuse
across the membrane and give rise to the highly reactive hydroxyl
radical. H
O
has been widely used to assess the
role of ROS in cells. To test whether H
O
directly added to the cells can activate MKK1/2, HeLa cells were
treated with 0.1-4 mM H
O
for 5
min or with 1 mM H
O
for 2.5-30
min, and the MKK1/2 activity was determined. Fig. 2shows that
H
O
caused a concentration- and time-dependent
activation of MKK1/2 in HeLa cells. Thus, H
O
alone is sufficient to induce MKK1/2 activation. The maximal activity
of MKK1/2 induced by H
O
in HeLa cells was
detected approximately 5 min after treatment. Thus, the kinetics of
H
O
induction is similar to that of MKK1/2
activation induced by phospholipids and growth
factors(13, 30) . However, 2 mM
H
O
, a concentration of H
O
that cannot be achieved in HeLa cells by LPA (data not shown), is
required to induce MKK1/2 activation to a similar magnitude
(approximately 20-fold) as 20 µM LPA.
Figure 2:
Activation of MKK1/2 by
HO
. Serum-deprived HeLa cells were treated
without (-ATZ) or with 50 mM aminotriazole
(+ATZ) for 60 min followed by stimulation for 5 min with
different concentrations of H
O
as indicated (A) or with 1 mM H
O
in the
absence of aminotriazole for the indicated time (B). Total
activity of MKK1/2 was determined. The phosphorylation of K52R was
quantitated with a PhosphorImager (Molecular Dynamics) after SDS-gel
electrophoresis. The basal activity (21 pmol/min/mg) of MKK1/2 in
unstimulated cells was arbitrarily set as 1
unit.
Several
possibilities exist that may account for the requirement of high
concentrations of HO
. First, the LPA-induced
ROS may be generated at a site that is more proximal to the target,
whereas the external added H
O
diffuses
indiscriminately. Second, HeLa cells may contain relatively high
catalase activity, and, thus, high concentrations of
H
O
are required to offset the catalase
activity. In fact, inhibition of catalase by preincubation of HeLa
cells with catalase inhibitor aminotriazole (26) prior to the
addition of H
O
resulted in a marked shift of
the H
O
dose-response curve to the left (Fig. 2A). However, even in the presence of
aminotriazole, greater than 0.5 mM H
O
was still required to activate MKK1/2 to a similar extent as that
induced by 20 µM LPA. Finally, it is likely that one or
more signaling events besides production of ROS are critical for the
induction of MKK1/2 activation by LPA, and ROS may function as only one
of the parallel signaling intermediates. Thus, although
H
O
alone at low concentrations (<0.5
mM) has a marginal effect on MKK1/2 activation,
H
O
and the derived radicals may have a greater
effect in the presence of other LPA-induced signaling intermediates
because of synergism.
To further confirm the involvement of ROS, we
treated HeLa cells with or without the catalase inhibitor aminotriazole
prior to LPA stimulation. If ROS participate in the LPA-stimulated
MKK1/2 activation pathway, inhibition of catalase would potentially
augment the response to LPA. A 4.5-fold increase in
HO
-induced MKK1/2 activity was observed when
HeLa cells were pretreated with aminotriazole (50 mM, 60 min),
demonstrating the effectiveness of the catalase inhibitor (Fig. 3, see also Fig. 2A). In cells pretreated
with aminotriazole, the LPA-stimulated MKK1/2 activity was 1.9-fold
that of cells without aminotriazole pretreatment (36.3- and 18.8-fold
above basal, respectively) (Fig. 3). Thus, decreasing the
catalase activity effectively enhances the cellular response to LPA,
indicating the involvement of ROS.
Figure 3:
Effect
of aminotriazole on the lysophosphatidic acid-stimulated MKK1/2
activation. MKK1/2 activity was determined in HeLa cells treated with
or without a catalase inhibitor aminotriazole (50 mM, 60 min)
and stimulated for 5 min with HO
(1
mM) or lysophosphatidic acid (20 µM). The kinase
reaction was carried at 30 °C for 10 min. The data represent the
average and range of two experiments.
For ROS to fulfill the role of
signaling intermediates for LPA, LPA must be able to induce the
production of ROS. As described above, LPA rapidly liberates
arachidonic acid in HeLa cells. Arachidonic acid is known to generate
ROS. Tumor necrosis factor and interleukin-1, both of which are
known to utilize ROS as signaling intermediates, also stimulate the
release of arachidonic acid(31) . However, other routes of ROS
generation are not excluded. We measured the relative concentrations of
H
O
in HeLa cells using dihydrorhodamine 123 and
fluorescence-activated cell sorting (FACS)(6, 24) .
Dihydrorhodamine 123 is oxidized to membrane-impermeable, fluorescent
rhodamine 123 in the presence of H
O
and
possibly ROS derived from it(24) . To minimize the loss of
H
O
, aminotriazole was also added to the media.
Incubation of HeLa cells with aminotriazole resulted in a
time-dependent increase in fluorescence intensity ( Fig. 4and
data not shown). A small increase of the fluorescence intensity induced
by LPA was detectable at the earliest time (5 min) examined, but more
consistent data were obtained if cells were stimulated for 10 min. In
two duplicated experiments, cells treated for 10 min with aminotriazole
(50 mM) plus BSA had an average 25% increase in mean
fluorescence intensity of rhodamine 123 compared with BSA-treated cells (Fig. 4). An additional 22% increase in mean fluorescence
intensity was detected in cells treated for 10 min with LPA (30
µM) plus aminotriazole (50 mM) (Fig. 4).
Thus, LPA is capable of generating ROS in HeLa cells.
Figure 4:
Relative levels of intracellular
HO
in HeLa cells. Serum-starved HeLa cells were
incubated for 20 min with 10 µM dihydrorhodamine 123,
followed by a 10-min incubation with either 50 mM aminotriazole plus BSA (B), 50 mM aminotriazole
plus 30 µM LPA (C), or an equivalent volume of
BSA (A). The fluorescence intensities of 10,000 cells were
analyzed.
In summary,
data presented in this study show that ROS are involved in the
LPA-induced MAP kinase kinase activation and LPA can stimulate the
production of ROS in HeLa cells. Additional experiments using a
p42 immune complex kinase assay (14) showed that N-acetylcysteine partially inhibited the LPA-stimulated
p42
activation, H
O
stimulated
p42
activity in HeLa and NIH 3T3 cells, and
aminotriazole enhanced the effect of LPA on p42
activation (data not shown). Previous studies have shown that ROS
are involved in the tumor necrosis factor
-stimulated NF-
B
activity (5) and the basic fibroblast growth factor-induced
c-fos expression(6) . Other data that we have obtained
showed that N-acetylcysteine also inhibited the LPA-stimulated
NF-
B and AP-1 DNA binding activities in HeLa cells. (
)Thus, ROS appear to function as signaling intermediates of
LPA and mediate a branch of the LPA signaling pathways. Our findings
lend support to the emerging concept that ROS can function as
physiological signaling intermediates. It will be interesting to
examine whether ROS also participate in the signaling pathways of other
phospholipids, such as platelet-activating factor (32) and
sphingosine 1-phosphate(33) . Clearly, further investigation of
the mechanisms by which LPA increases the intracellular levels of ROS
and ROS relay the cellular regulatory signals is warranted.