(Received for publication, July 20, 1994; and in revised form, November 15, 1994 )
From the
Basic fibroblast growth factor (FGF) stimulates the
proliferation, differentiation, and motility of multiple cell types.
Signal transduction by FGF is mediated by high affinity FGF receptors
that have autophosphorylating tyrosine kinase activity and also elicit
the release of low molecular weight signaling molecules, including
inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have
shown previously that basic FGF-stimulated, phospholipase A (PLA
)-mediated arachidonate release regulates
endothelial cell (EC) motility (Sa, G., and Fox, P. L.(1994) J.
Biol. Chem. 269, 3219-3225). Here we identify the
phospholipase responsible for basic FGF-mediated arachidonate release
as cytosolic PLA
(cPLA
) by demonstrating in EC
lysates a requirement for micromolar Ca
,
dithiothreitol insensitivity, and inactivation by anti-cPLA
antiserum. The role of cPLA
is also indicated by the
observed mechanisms of activation which show a requirement for p42
mitogen-activated protein kinase activity, cPLA
phosphorylation, and cPLA
translocation from cytosol
to membranes. Phosphorylation of cPLA
, arachidonate release
from prelabeled intact cells, and cell motility all have similar
concentration dependencies on basic FGF. Since arachidonate release is
required for basic FGF-stimulated motility of EC, our results show that
p42 mitogen-activated protein kinase activation of cPLA
may
be a regulatory event in stimulation of cellular release of this
important eicosanoid precursor during cellular responses to basic FGF.
Basic fibroblast growth factor (FGF) ()is a potent
mitogenic and chemotactic factor for a variety of cells. Its activity
has been implicated in multiple physiological and pathological
processes including differentiation, wound healing, blood vessel
intimal hyperplasia, and tumor angiogenesis(1) . The cellular
effects of basic FGF are transduced by its interaction with any one of
four members of a family of high affinity, cell surface FGF
receptors(2) . These receptors are ``single-pass''
transmembrane proteins with a kinase activity that induces
phosphorylation of tyrosine residues in the receptor itself, as well as
in phospholipase-C
1(3) , a 34-kDa lipocortin-like
protein(4) , cortactin(5) , and Shc(6) . Of
these proteins only phospholipase-C
1 is known to be a direct
substrate of the FGF receptor tyrosine kinase. Basic FGF also triggers
a series of downstream events, including activation of p21
(7) and mitogen-activated protein (MAP) kinases (8) and expression of early response genes(9) . In
addition, basic FGF rapidly induces the release of arachidonate from
bovine aortic endothelial cells (EC) (10, 11) and from
Swiss 3T3 cells(12) .
Eicosanoids derived from arachidonate
elicit multiple physiologic and pathophysiologic responses. We have
shown that arachidonate release is necessary for basic FGF-stimulated
EC movement(10) . The release appears to be coupled to a
pertussis toxin-sensitive G-protein which is required for EC migration
but not for proliferation. Arachidonate release may thus be responsible
for divergent signaling pathways initiated by the FGF receptor. The
enzymatic activity responsible for arachidonate release during basic
FGF-mediated events has not been established, nor have the mechanisms
of activation been investigated. The arachidonate released does not
appear to be derived from diacylglycerol generated by
phosphoinositidedependent phospholipase C(11, 12) . We
and others have shown that phospholipase A (PLA
) inhibitors, albeit of uncertain specificity,
almost completely block basic FGF-stimulated arachidonate
release(10, 12) . Direct measurement of PLA
activity by an ``in vitro'' assay of EC
lysates using L-
-1-palmitoyl-2-[1-
C]arachidonyl
phosphatidylcholine (PC) as substrate has shown that basic FGF
stimulates PLA
activity by about 80%(10) .
At
least two distinct forms of PLA are present in most
mammalian cells (see (13) for review). A low molecular mass
(14 kDa) enzyme, dependent on a high concentration (mM) of
Ca
(secretory PLA
) is homologous to forms
secreted by the pancreas. A second form, cytosolic PLA
(cPLA
), has been recently identified and purified
from human monocytic cells (14) and cloned from mRNA isolated
from U937 cells(15, 16) . Cytosolic PLA
is
characterized by a high molecular mass (85-100 kDa), activation
by a low concentration (µM) of Ca
,
selectivity for arachidonate in the sn-2 position of
phospholipids, and insensitivity to disulfide reducing
agents(14, 17) . Recent studies have shown that
cPLA
is activated by phosphorylation by both protein kinase
C and by p42 MAP kinase(18, 19, 20) . The
phosphorylated enzyme is translocated from the cytosol to the plasma
membrane in a process utilizing a Ca
-dependent
phospholipid-binding domain in the N-terminal region of
cPLA
(15) .
In this report we show that the
PLA activated by basic FGF is cPLA
, that the
activation occurs by a MAP kinase-dependent phosphorylation pathway,
and that this activity is responsible for the release of arachidonate
in stimulated EC.
Figure 1:
Phospholipase A activity in EC fractions. A, cytosolic and membrane
fractions were prepared from EC lysates by ultracentrifugation and
Percoll gradient, respectively. The fractions were treated with (solid bars) or without (striped bars) 10 ng/ml of
basic FGF for 15 min. PLA
activity was measured by
hydrolysis of L-
-1-palmitoyl-2-[
C]arachidonyl
PC. B, same as A except that EC were pretreated in
the presence (solid bars) or absence (striped bars)
of 10 ng/ml of basic FGF before preparation of the cytosolic and
membrane fractions.
Figure 2:
Ca dependence of basic
FGF-stimulated PLA
activity in EC lysate. Membrane and
cytosol fractions were prepared from semi-confluent EC.
Ca
-depleted cytosol was prepared by incubation with
Chelex 100 resin and combined with the particulate membrane fraction in
the presence (
) or absence (
) of 10 ng/ml basic FGF for 15
min. PLA
activity in the mixture was determined by
hydrolysis of L-
-1-palmitoyl-2-[
C]arachidonyl PC
in the presence of various concentrations of exogenous
Ca
. The difference is the activity due to basic FGF
(
). The data were fitted by weighted, nonlinear least squares
regression using the method of Levenberg-Marquardt(28) ; the
two-enzyme Michaelis-Menten equation was used for basal and stimulated
activities and the one-enzyme equation for the
difference.
The effect of a rabbit anti antibody directed against human
cPLA on the activity of EC lysates was tested using the in vitro PLA
assay. The antibody blocked
essentially all basic FGF-stimulated activity, confirming that the
PLA
activated by basic FGF in EC is cPLA
(Fig. 3). The activity of purified cPLA
is
insensitive to reduction by dithiothreitol(14) ; likewise, the
basic FGF-stimulated PLA
activity in EC lysates was found
to be insensitive to dithiothreitol (<3% inhibition, not shown)
providing further confirmation of the role of cPLA
.
Figure 3:
Inhibition of basic FGF-stimulated
arachidonate release by anti-cPLA. An EC lysate was
incubated in the presence of rabbit anti-cPLA
antiserum (or
control serum) for 1 h at 4 °C before assay. Phospholipase A
activity was measured as the release of
[
C]arachidonate in the presence (solid
bars) or absence (striped bars) of 10 ng/ml basic
FGF.
Figure 4:
Role of
p42 MAP kinase-mediated phosphorylation in basic FGF-stimulated
PLA activity. Membrane and cytosol fractions were prepared
from lysates of semi-confluent EC as described in the legend to Fig. 1. p42 MAP kinase was immunodepleted with rabbit anti-p42
MAP kinase antiserum (or control serum). Immunodepleted cytosol was
combined with the membrane fraction in the presence (striped
bar) or absence (solid bar) of 10 ng/ml basic FGF.
PLA
activity of the combined fractions was determined by
hydrolysis of L-
-1-palmitoyl-2-[
C]arachidonyl
PC. The combined fractions were also incubated with calf intestinal
phosphatase (20 µg/ml) for 30 min at 37 °C before measurement
of PLA
activity.
Figure 5:
Time course of basic FGF-stimulated
phosphorylation of p42 MAP kinase and cPLA in EC. A, semi-confluent EC cultures (25
10
cells) were prelabeled for 3 h with
[
P]orthophosphate (300 µCi/ml) and then
stimulated with basic FGF (10 ng/ml) for up to 20 min. The cells were
lysed by sonication, immune complexes formed by addition of polyclonal
anti-p42 MAP kinase antiserum, and the immunoprecipitates subjected to
SDS-PAGE and autoradiography. B, semi-confluent EC cultures
(25
10
cells) were incubated with 10 ng/ml of basic
FGF at 37 °C for the time indicated. Cell lysates were subjected to
SDS-PAGE and immunoblotted with rabbit anti-human cPLA
. C, phosphorylation of cPLA
(
) and p42 MAP
kinase (
) were quantitated by densitometry and expressed as the
percent of total immunodetectable cPLA
and relative
densitometric units, respectively.
Figure 6:
Concentration dependence of cPLA phosphorylation in basic FGF-stimulated EC. Semi-confluent bovine
aortic EC (25
10
cells) were incubated with various
concentrations of basic FGF for 12 min at 37 °C. The cells were
lysed, subjected to SDS-PAGE electrophoresis, transferred to
polyvinylidene difluoride membrane, and immunoblotted with
anti-cPLA
antibody. The positions of unphosphorylated and
phosphorylated cPLA
(cPLA
-P) are indicated by large arrows.
Activation of cPLA requires not only
phosphorylation but also Ca
-dependent translocation
of the enzyme from cell cytoplasm to
membranes(15, 18, 20, 30, 31) .
The experiments in Fig. 1suggested that translocation of
PLA
activity occurred in response to basic FGF treatment of
cell extracts or living cells. To specifically demonstrate cPLA
translocation, membrane and cytosolic fractions from basic
FGF-treated EC were isolated and subjected to SDS-PAGE and
immunoblotting with anti-cPLA
antibody (Fig. 7).
These data clearly show that nearly all cPLA
is
translocated to membranes within 12 min after stimulation of EC with
basic FGF.
Figure 7:
Basic FGF-stimulated translocation of
cPLA from cytosolic to membrane fractions. Semi-confluent
EC cultures (25
10
cells) were incubated with 10
ng/ml of basic FGF at 37 °C for 12 min. Lysates were prepared and
cytosolic (Cyto.) and membrane (Mem.) fractions were
isolated by ultracentrifugation and Percoll gradient, respectively. The
fractions were subjected to SDS-PAGE and immunoblotted with rabbit
anti-human cPLA
.
The in vitro
PLA assay results suggested that essentially all basic
FGF-stimulated PLA
activity was due to activation of
cPLA
. In a dose-response experiment, maximal stimulation of
basic FGF-induced [
H]arachidonate release was
half-maximal at about 0.3 ng/ml of the growth factor with 80%
stimulation at maximal basic FGF concentration; the concentration
dependence was similar to that of cPLA
phosphorylation (Fig. 8). This result is consistent with the observation that
complete phosphorylation of cPLA
results in 2-fold
stimulation of specific activity(32) . We showed previously
that arachidonate release was required for basic FGF-stimulated EC
motility (10) . The concentration dependence of phosphorylation
of cPLA
and release of arachidonate were similar to that of
basic FGF-stimulated EC movement (Fig. 8). In fact, the
stimulation of arachidonate release and cell movement were essentially
identical. These data, and the above results showing that essentially
all basic FGF-stimulated PLA
activity is due to
cPLA
, suggest that cPLA
activity has functional
cellular consequences and may be a critical factor regulating EC
motility.
Figure 8:
The effect of basic FGF on phosphorylation
of cPLA, [
H]arachidonate release, and
EC migration. Bovine aortic EC were prelabeled by incubation with
[
H]arachidonate (0.5 µCi/ml) for 24 h. The
cells were then stimulated with basic FGF for 15 min and release of
[
H]arachidonate into the medium was measured
(- - -
- - -); unstimulated
[
H]arachidonate release was 32.3 fmol/well. Basic
FGF-stimulated phosphorylation of cPLA
was quantitated by
densitometry of the slow moving band in the Western blot shown in Fig. 6(-
-, relative
densitometric units). Cell migration was measured as the number of
cells crossing the wound line during a 22-h incubation (- -
-
- - -); the number of migrating cells in
the absence of exogenous basic FGF was 226/3000 µm of wound edge.
All data were fitted to the one-enzyme Michaelis-Menten equation by
nonlinear least squares regression using the method of
Levenberg-Marquardt(28) .
Agonist-induced release of arachidonate, and the subsequent
conversion to bioactive eicosanoids, regulates multiple normal and
pathological EC processes(33) . Basic FGF has been shown to
stimulate arachidonate release in EC, but the enzymes involved and the
mechanism(s) underlying the activation have not been
defined(10, 11) . In this report we provide evidence
that basic FGF stimulates the activity of the cytosolic form of
PLA. The activated enzyme has biochemical and enzymatic
characteristics consistent with cPLA
, namely, a requirement
for µM Ca
and dithiothreitol
insensitivity. Furthermore, basic FGF-stimulated PLA
activity in EC lysates is almost completely blocked by
cPLA
-specific antiserum. The mechanism of activation is
also consistent with cPLA
, namely, p42 MAP kinase activity
is required, and enzyme translocation from the cytosol to membranes
occurs. Together these data represent convincing evidence that basic
FGF activates cPLA
in EC.
Basic FGF is far from unique
in its activation of cPLA, but it has characteristics that
distinguish it from several other agonists. We have shown, by antibody
inactivation and by Ca
titration, that essentially
all basic FGF-induced PLA
activity is due to activation of
cPLA
. This specificity is not universal, for example,
anti-cPLA
antibody inhibits only half of the PLA
activity in lysates of human umbilical vein EC stimulated by
tumor necrosis factor, the remaining activity is contributed by the
secretory form(34) . Platelet-derived growth factor may also
stimulate both cPLA
as well as a distinct, unidentified
PLA
(35) . We have also found that basic FGF by
itself stimulates PLA
activity not only in lysates, but
also when measured as release of arachidonate by intact cells. This
observation qualitatively differentiates the activation of cells by
basic FGF from that by vasopressin, epidermal growth factor, and
phorbol esters, which stimulate cPLA
activity in extracts
without significantly increasing cellular release of
arachidonate(35) . Similarly, granulocyte/macrophage
colony-stimulating factor (31) and tumor necrosis factor (36) only minimally activate cellular arachidonate release in
the absence of agents that increase calcium transport.
The induction
by basic FGF is much less than the 20-fold stimulation of arachidonate
release induced by platelet-derived growth factor in 3T3
cells(35) . The mechanisms underlying agonist activation of
cellular arachidonate release are only partly understood, but the
ability to mobilize calcium may be one critical component. Previous
observations showing that basic FGF stimulates Ca flux (37) are consistent with this mechanism. The finding
that platelet-derived growth factor stimulates calcium transients more
effectively than basic FGF may explain the relative abilities of these
agonists to stimulate cPLA
(38) . Since thrombin
mobilizes EC calcium fluxes (39) , our finding that it is an
ineffective agonist of PLA
suggests that calcium alone is
not sufficient; this result confirms a previous observation in which EC
prostacyclin synthesis is not induced by thrombin(40) .
The
observation that FGF receptor stimulation is sufficient to activate
cPLA and cause intracellular release of arachidonate even
in the absence of auxiliary factors (such as calcium ionophores) is
consistent with the pluripotent activity of the receptor(2) .
Our data, and that of others, indicate the requirement for p42 MAP
kinase for activation of cPLA
. This observation is also
consistent with the known activation of MAP kinases by basic (8) and acidic (6) FGF, most likely by a
p21
-dependent
pathway(6, 7, 41, 42) . However,
activation of MAP kinase alone may not be sufficient to fully activate
cPLA
, since granulocyte/macrophage colony-stimulating
factor activates MAP kinase, but is a rather weak activator of
cPLA
(31) . An additional activity required for full
activation of cPLA
may be protein kinase C, which activates
cPLA
by MAP kinase-dependent and -independent
mechanisms(18, 19, 20) . Activation of this
kinase by basic FGF and its role in FGF-stimulated cell movement and
proliferation have been reported(43) . The role of
Ca
in translocation and activation of cPLA
has been clearly shown. Diacylglycerol-mediated activation of
protein kinase C and IP
-mediated mobilization of
intracellular Ca
may both result from activation of
phospholipase C-
1 by the FGF receptor. Interestingly, FGF-induced
tyrosine phosphorylation and activation of phospholipase C-
1 is
not required for FGF receptor-mediated cell proliferation(44) ,
PC12 cell differentiation (6) , and chemotaxis(45) ,
but its role in activation of cPLA
is not known. Finally,
there is evidence for a role of guanine nucleotide-binding proteins
(G-protein) in activation of PLA
(46) , and most
likely, cPLA
(47, 48) (but
G-protein-independent activation of cPLA
has also been
reported(49) ). We and others (10, 50, 51) have shown that several basic
FGF-dependent processes depend on pertussis toxin-sensitive G-proteins
and that basic FGF-stimulated arachidonate release in EC is completely
blocked by the toxin(10) .
The activation of multiple signal
transduction pathways by the FGF receptor is most likely responsible
for its sufficiency to stimulate functional cPLA activity.
The identification of cPLA
-coupled G-proteins, the precise
mechanisms by which these and other pathway components regulate
cPLA
activity, and the role that cPLA
has in
regulation of EC motility and other cellular functions are important
questions that require further investigation.