Department of Biological Sciences, Allergan, Incorporated, Irvine, California 92612
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although both
vascular endothelial growth factor (VEGF) and fibroblast growth factor
(FGF) receptors have been shown to be important in the regulation of
vascular endothelial cell growth, the roles of phospholipase C (PLC)
and Ca2+ in their downstream signaling cascades are still
not clear. We have examined the effects of VEGF and FGF on PLC
phosphorylation and on changes in intracellular Ca2+ levels
in primary endothelial cells. VEGF stimulation leads to PLC
activation and increases in intracellular Ca2+, which are
correlated with mitogen-activated protein (MAP) kinase (MAPK)
activation and cell growth. Inhibition of Ca2+ increases by
the Ca2+ chelator
1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM resulted in marked inhibition of MAPK activation, which was
shown to be linked to regulation of cell growth in these cells. In
contrast, FGF stimulation did not lead to PLC
activation or to
changes in intracellular Ca2+ levels, although MAPK
phosphorylation and stimulation of cell proliferation were observed.
Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on these
FGF-stimulated responses. These data demonstrate a direct role for
PLC
and Ca2+ in VEGF-regulated endothelial cell growth,
whereas this signaling pathway is not linked to FGF-mediated effects in
primary endothelial cells. Thus endothelial cell-specific factors
regulate the ability of VEGF receptors and FGF receptors to couple to
this signaling pathway.
vascular endothelial cell growth factor; fibroblast growth factor; phospholipase C; calcium; choroidal endothelial cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ANGIOGENESIS, the formation of new capillaries from preexisting blood vessels, is characteristic of a number of pathological processes, including tumor growth and ocular neovascularization. It is a complex response dependent on the interaction of a variety of factors, including cytokines, proteases, adhesion molecules, and inflammatory mediators (34, 37). Among the better-characterized are growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which have been shown to regulate vascular endothelial cell growth, migration, and differentiation. Both VEGF and bFGF are known to mediate their effects through stimulation of cell surface receptors that are members of the tyrosine kinase receptor family. The VEGF receptors comprise three members (VEGFR-1 through -3) and belong to the platelet-derived growth factor receptor subfamily of receptor tyrosine kinases (RTK) (24, 36, 51). All VEGF receptors are expressed early in development and are mostly confined to endothelial cells. In adults, VEGFR-1 has also been detected in macrophages and monocytes in addition to endothelial cells, whereas VEGFR-3 is largely restricted to lymphatic endothelium. VEGFR-2 expression is limited primarily to endothelial cells and is believed to mediate most of the biological effects of VEGF including mitogenesis (36, 51).
The FGF receptor (FGFR) family is composed of four gene products, FGFR-1 through -4, and these genes give rise, through alternative splicing, to a number of receptor subtypes (40). These receptors are widely expressed in multiple-organ systems, including vascular endothelium, suggesting functional roles in a number of homeostatic mechanisms (15). In contrast to VEGF, which is an endothelial cell-specific mitogen, FGF acts on a variety of different cell types, functioning as both a direct and an indirect stimulator of angiogenesis. How VEGF and FGF work together to modulate the overall response is not clear, although they have been shown to act synergistically to promote angiogenic responses both in vitro and in vivo (26, 33).
The activation of RTK is dependent on receptor dimerization and
autophosphorylation of tyrosine residues in the cytoplasmic domain of
the receptor (14, 38). Phosphorylation of these tyrosine
residues serves to activate the enzymatic activity of the receptor
(11, 16) as well as to provide binding sites for enzymes
and adaptor proteins that are important in downstream signaling events.
Stimulation of VEGF receptors has been shown to lead to the activation
of phospholipase C (PLC) (7, 12), phosphatidylinositol
3-kinase (12, 43, 50), and Akt (29). Whether
these early signaling events are mediated through direct binding to the
receptor or through the activity of adaptor proteins such as the
recently described VEGFR adaptor protein (VRAP) (48) is
not clear. Activation of FGF receptors, on the other hand, has been
shown to lead to direct binding and phosphorylation of PLC
(28) and to the binding of FRS-2 and associated proteins (20, 22). For both VEGF and FGF receptors, these early
activation events are linked to downstream signaling pathways that
regulate cellular responses such as proliferation, migration, and the
synthesis and release of cell signaling molecules.
It has been demonstrated that VEGF and bFGF induce cell proliferation
through a mitogen-activated protein (MAP) kinase (MAPK)-dependent pathway in endothelial cells (13, 25). What is not known
is how effector proteins coupled to the VEGF and FGF receptors are linked to the MAPK pathway and how they contribute to the overall growth response. In addition to PLC phosphorylation, the production of inositol 1,4,5-trisphosphate (IP3) and increased protein
kinase C (PKC) activity have been shown to be associated with VEGFR
activation (2, 12, 49). This is consistent with
experiments that directly measured VEGF-induced increases in
intracellular Ca2+ levels in endothelial cells
(2). Although PLC
phosphorylation and increases in
intracellular Ca2+ levels in response to FGF stimulation in
fibroblasts and in cells overexpressing FGFR have been reported
(3, 35, 44), characterization of the role of this pathway
in endothelial cells expressing endogenous FGFR has not been
established. Furthermore, although PLC
has been implicated in the
mitogenic response induced by a number of growth factors, its role in
mediating VEGF- and FGF-induced proliferation in endothelial cells is
unclear. It was the purpose of this study to examine the role of PLC
and Ca2+ in regulating the MAPK pathway and cell
proliferation in primary endothelial cells in response to VEGF and FGF.
We have chosen choroidal endothelial cells (CEC) because they are known
to express both VEGF and FGF receptors (27, 45) and have
been shown to be regulated by both FGF and VEGF in ocular angiogenic
responses (19, 23). Our studies demonstrate that both VEGF
and FGF receptor activation lead to MAPK stimulation and a subsequent
increase in CEC proliferation. Phosphorylation of PLC
was found to
occur through stimulation of VEGFR but not FGFR, consistent with the observed increase in intracellular Ca2+ levels in response
to VEGF but not FGF in these cells. Furthermore, inhibition of the rise
in intracellular Ca2+ induced by VEGF led to inhibition of
MAPK activation, whereas inhibition of PLC
activity led to a
reduction in cell growth in VEGF- but not FGF-stimulated cells. To the
best of our knowledge, these data demonstrate for the first time a
distinct difference in the role of PLC
and Ca2+ in
regulating cell growth by directly comparing VEGF- and FGF-stimulated primary endothelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Recombinant human VEGF 165 and recombinant human bFGF were obtained from R&D Systems (Minneapolis, MN). U-73122 was purchased from Biomol (Plymouth Meeting, PA) and PD-98059 from Calbiochem (San Diego, CA). 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was obtained from Molecular Probes (Eugene, OR).
Antibodies.
Monoclonal antibody specific to phosphotyrosine and mixed monoclonal
antibodies to PLC were obtained from Upstate Biotechnology (Lake
Placid, NY). The rabbit polyclonal anti-Flk-1 antibody directed against
an epitope in the carboxy terminus of the receptor was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies to
phospho-p44/p42 MAPK and polyclonal antibodies to p44/42 MAPK were from
New England Biolabs (Beverly, MA). FGFR-1-specific polyclonal antiserum
was a gift from Harald App (Sugen, South San Francisco, CA).
Cell culture. CEC, isolated from bovine eyes as described previously (30), were cultured in supplemented Iscove's modified Dulbecco's medium containing 10% human serum on collagen IV-fibronectin-laminin-coated tissue culture plates. Cells from passages 2-5 were used for all experiments. For growth factor treatments, subconfluent cells were stimulated with 10 ng/ml VEGF or 10 ng/ml bFGF for the indicated time periods.
Detection of endogenous RTK autophosphorylation, PLC substrate
phosphorylation, and MAPK activation.
Subconfluent CEC were starved in media containing 1% FBS overnight
followed by stimulation with growth factors for 5 min at 37°C. For
measurement of VEGF receptor autophosphorylation and MAPK
phosphorylation, the reaction was stopped by the addition of boiling
Laemmli sample buffer. To test the effects of BAPTA-AM and PD-98059 on
MAPK activation, cells were pretreated with agent for 30 min before
growth factor stimulation. Equal amounts of whole cell protein lysates
were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and
analyzed by Western blotting. To measure growth factor-induced PLC
tyrosine phosphorylation and FGFR autophosphorylation, cells were
treated with growth factor as above and lysed with Triton lysis buffer
(50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1%
Triton X-100, 1 µg/ml leupeptin, pepstatin A, and aprotinin, 1 mM
phenylmethysulfonyl fluoride, and 1 mM sodium orthovanadate). PLC
and FGFR were immunoprecipitated and immunoblotted to determine
phosphorylated tyrosine levels as described in
Immunoprecipitation and immunoblotting.
Immunoprecipitation and immunoblotting. For immunoprecipitation, cell lysates were incubated with primary antibody and immunocomplexes were collected by further incubating the lysates with protein G-agarose beads. The immunoprecipitates were washed with lysis buffer, resuspended in Laemmli sample buffer, and analyzed by immunoblotting. For immunoblotting, equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose. After transfer, the membranes were blocked in phosphate-buffered saline, pH 7.2, containing 0.1% Tween 20 (PBST) and 5% bovine serum albumin for 1 h. Membranes were then probed with primary antibody for 2 h. After extensive washes in PBST, immunoreactive proteins were visualized by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies (BioRad) and ECL reagent (Amersham).
Assay of DNA synthesis. CEC were plated onto 96-well fibronectin-coated plates at a subconfluent density of 1,000 cells/well in complete growth media. After 24 h, the cells were pretreated with inhibitor for 30 min at 37°C followed by incubation with growth factor for 48 h in medium containing 0.5% FBS. Enzyme immunoassay was used for the determination of cell proliferation by quantification of 5-bromo-2'-deoxyuridine incorporation into DNA (Roche Molecular Biochemicals).
Calcium measurements. CEC were loaded with 2 µM fura 2-AM in suspension for 30 min at 37°C in basal media. Cells were washed in HEPES buffer and divided into 1-ml aliquots containing 1 × 106 cells. Changes in intracellular Ca2+ concentration were measured on a Perkin Elmer luminescence spectrometer. Fluorescence was measured at 340 and 380 nm, and intracellular free calcium was calculated using the 340-to-380 ratio. Maximum and minimum fluorescence were determined by lysing the cells with digitonin and EGTA calcium chelation, respectively. For each run, a stable baseline fluorescence was achieved followed by stimulation with growth factors. To determine the effect of BAPTA-AM on VEGF-induced Ca 2+ signaling, various concentrations of BAPTA were incubated with the cells for 30 min before VEGF stimulation.
Statistical analysis. All values are expressed as means ± SE obtained from three independent experiments. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni t-test. P values <0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VEGF and FGF induce RTK autophosphorylation in CEC.
Activation of intrinsic kinase activity in response to growth factor
binding is the initial step in signal transduction by RTK leading to a
biological response. Receptor autophosphorylation in response to VEGF
and FGF has been characterized both in endothelial cells expressing
endogenous receptors and in nonendothelial cell types expressing
recombinant receptors. The ability of VEGF and FGF to stimulate
receptor autophosphorylation in bovine CEC was confirmed by Western
blot analysis (Fig. 1). A
tyrosine-phosphorylated protein of ~230 kDa was identified in lysates
from CEC stimulated with VEGF but not in nonstimulated control cells
(Fig. 1A). This phosphorylated protein corresponded to
VEGFR-2, as determined by reprobing the blot with anti-flk
antibody (Fig. 1A). Similarly, when FGFR-1 was
immunoprecipitated, the phosphorylated form of this receptor was
identified only in lysates from FGF-stimulated cells (Fig.
1B). Three isoforms of the FGF receptor were
immunoprecipitated from both control and stimulated lysates as
identified by immunoblotting with anti-FGFR-1 antibodies; however, only
the 116-kDa form appears to be phosphorylated in response to FGF
stimulation (Fig. 1B). Time course and
concentration-response experiments determined that maximal stimulation
occurred at 10 ng/ml after 5 min of stimulation for both growth factors
(data not shown). These data confirm VEGFR-2 and FGFR-1
autophosphorylation in response to their respective ligands in bovine
CEC.
|
Activation of MAPK by VEGF and bFGF.
The MAPK cascade is a common signaling pathway coupled to mitogenesis
in many cell types. Because we were interested in the pathways
mediating VEGF and FGF proliferation in CEC, we next determined the
effects of these growth factors on MAPK activation. After overnight
starvation, CEC were stimulated with 10 ng/ml VEGF or bFGF over a 1- to
15-min period. Lysates were analyzed by Western blot using a
phospho-specific p44/42 MAPK monoclonal antibody. Maximum MAPK
activation was found to occur within 5-10 min after VEGF or bFGF
addition (Fig. 2A).
Furthermore, the response to both growth factors was transient,
returning to baseline levels within 60 min (data not shown). Previous
studies showed MAPK activation to be dependent on the raf/MAPK kinase
(MEK) signaling cascade (1, 40, 51). We therefore
looked at the effect of the MEK1 inhibitor PD-98059 on MAPK
phosphorylation (Fig. 2B). Complete inhibition of both VEGF
and bFGF MAPK activation was observed in the presence of 10 µM
PD-98059. Compared with VEGF, activation of MAPK by bFGF appeared to be
more sensitive to inhibition of MEK1. These data demonstrate a role for
MEK1 in the induction of MAPK activity by VEGF and bFGF.
|
MAPK cascade couples VEGF and bFGF receptor activation to
proliferation in CEC.
VEGF and bFGF are known to stimulate cell growth in different
endothelial cell types. Similarly, in CEC VEGF and bFGF produced a
concentration-dependent induction of DNA synthesis (Fig.
3A). PD-98059 produced
significant inhibition of VEGF growth at ~10 µM, consistent with
its ability to block MAPK activation (Figs. 2B and
3B). PD-98059 also reduced bFGF-stimulated DNA synthesis, again consistent with its inhibition of FGF-stimulated MAPK
phosphorylation, although it was less potent than against the
VEGF-driven response. Fifty micromolar PD-98059 was required to produce
80% inhibition of bFGF-stimulated growth, whereas ten micromolar
PD-98059 produced approximately the same effect against VEGF-stimulated
growth. The fact that significantly higher concentrations of
PD-98059 were required to inhibit bFGF-stimulated growth could be
explained by the fact that FGF is a stronger endothelial cell mitogen
than VEGF. Previous studies have shown that PD-98059 is less active against potent agonists such as epidermal growth factor or in cells
expressing high numbers of receptors (1). Our data
demonstrate that both VEGF- and FGF-driven proliferation are dependent
on MAPK activation.
|
Role of PLC/Ca2+ pathway in VEGF-
and bFGF-mediated proliferation.
PLC
has been shown to be a substrate for both VEGF- and
bFGF-activated receptors. Activation of this enzyme has been linked to
the production of IP3 and diacylglycerol leading to PKC
activation and increased intracellular Ca2+ levels. As
expected, VEGF, over a concentration range of 1-50 ng/ml,
stimulated Ca2+ mobilization in CEC. As shown in Fig.
4, VEGF (10 ng/ml) produced a response
that was characterized by an initial transient peak in Ca2+
followed by a sustained phase in which Ca2+ remained
elevated above baseline levels throughout the observation period. In
contrast, 10 ng/ml of bFGF failed to induce an increase in
intracellular Ca2+ levels compared with control in CEC
(Fig. 4). The upward drift of baseline in control and FGF-stimulated
cells is attributed to dye leakage over the 10-min observation period.
bFGF failed to elicit a Ca2+ signal at concentrations up to
50 ng/ml (data not shown).
|
|
Role of Ca2+ in VEGF- and
bFGF-induced activation of MAPK.
To determine whether PLC/Ca2+ signaling plays a direct
role in VEGF-mediated mitogenesis, we examined the effects of the
intracellular Ca2+ chelator BAPTA-AM on MAPK activation. We
first confirmed the direct effects of BAPTA-AM on intracellular
Ca2+ levels in response to VEGF. CEC were preincubated with
increasing concentrations of BAPTA-AM, over a range of 1-25 µM,
30 min before VEGF stimulation. It was observed that 25 µM BAPTA-AM
completely abolished VEGF-mediated Ca2+ mobilization (Fig.
6A). Because activation of
MAPK can occur through both Ca2+-dependent and
Ca2+-independent mechanisms (4), we next
determined whether VEGF-induced MAPK activation is effected by
chelating intracellular Ca2+. Incubating CEC with
increasing BAPTA-AM concentrations produced an inhibition of
VEGF-stimulated MAPK phosphorylation. Significant inhibition was
observed in the presence of 25 µM BAPTA (lower BAPTA concentrations
had no effect), which is consistent with the concentration requirements
for complete inhibition of intracellular Ca2+ levels (Fig.
6B). In keeping with our observation that FGF does not
elicit a Ca2+ signal in these cells, BAPTA-AM was shown to
have no effect on FGF-stimulated MAPK phosphorylation (Fig.
6B). BAPTA could not be tested directly for effects on
proliferation because of the toxic effects of long-term exposure in
CEC. These data support a role for PLC
and Ca2+ in the
signaling mediating proliferation through VEGF but not FGF receptors in
CEC.
|
PLC inhibitor U-73122 blocks VEGF- but not FGF-induced DNA
synthesis.
U-73122, a selective PLC inhibitor (39), was used to
determine whether the differential mechanisms implicated in VEGFR- and
FGFR-driven mitogenic pathways in fact play a role in regulating the
functional response in CEC. Inhibition of VEGF- or bFGF-stimulated DNA
synthesis by U-73122 was assessed in CEC cultures (Fig.
7). For these experiments, the highest
concentration of U-73122 that did not produce a cytotoxic effect on the
cells was used. VEGF- but not bFGF-stimulated growth was inhibited by
U-73122 in a concentration-dependent manner. One micromolar U-73122
produced an 80% inhibition of VEGF effects but no significant
inhibition of bFGF-stimulated growth (Fig. 7). These results confirm a
requirement for PLC in the VEGF-induced mitogenic response in
endothelial cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies identified components of the signal transduction
pathways activated by VEGF and bFGF in vascular endothelial cells and
linked them to functional responses such as migration and cell growth
(24, 36, 40). We were interested in regulation of
the MAPK cascade by VEGF and FGF receptors in endothelial cells and the
roles of PLC and Ca2+ in these pathways. We have
confirmed the presence of the early phosphorylation events, including
receptor autophosphorylation and MAPK activation, associated with
stimulation of endogenous VEGF and FGF receptors by their respective
ligands in bovine CEC. The kinetics of these responses are similar to
what was previously reported for bovine brain capillary endothelial
cells (9). In addition, the downstream activation of MAPK
was found to be sensitive to inhibition by the MEK inhibitor PD-98059,
which is in keeping with previous findings in primary endothelial cells (42, 47). Furthermore, PD-98059 is shown to inhibit DNA
synthesis driven by both VEGF and FGF in these cells. Thus our data
support a link between tyrosine kinase receptor phosphorylation, MAPK activation, and cell proliferation in response to both VEGF and FGF in CEC.
Regulation of the MAPK cascade and endothelial cell proliferation by
PLC has been inferred from previous studies. Xia et al.
(49) reported that in porcine aortic endothelial cells, VEGF stimulation led to PLC
tyrosine phosphorylation, inositol phosphate production, and cell proliferation. Similarly, in human umbilical vein endothelial cells (HUVEC), activated VEGFR-2 has been
shown to bind to and promote the phosphorylation of PLC
, which is
correlated with MAPK activation and increased 3H-labeled
thymidine incorporation into DNA (47). It has been argued,
in fact, that the PLC
/PKC pathway is the main regulator of the
VEGF-driven Raf/MEK/MAPK cascade in endothelial cells and that this is
a Ras-independent process (10, 42). Activation of the FGF
receptor in porcine aortic endothelial cells expressing the wild-type
receptor also leads to stimulation of PLC
activity. However, in
cells expressing a mutant receptor that fails to stimulate PLC
, MAPK
activation and cell proliferation are not effected (5).
VEGF-induced increases in intracellular Ca2+ are well known
(46) and are shown to be mediated through activation of
VEGFR-2 (8). Although the ability of FGF to induce
Ca2+ mobilization in fibroblasts and other cell types has
been reported (35, 44) and observed in our own laboratory,
the role of FGF in Ca2+ signaling in endothelial cells is
less clear. These observations notwithstanding, the roles of PLC
activation and the subsequent changes in intracellular Ca2+
levels mediated by VEGF and FGF receptors in the activation of MAPK and
the regulation of cell growth have not been examined directly. Our data
demonstrate a marked elevation of Ca2+ in response to VEGF
in CEC but no measurable change in response to FGF. This is consistent
with the observed stimulation of PLC
phosphorylation by VEGF but not
FGF. It is unlikely that the lack of response in CEC is due to
experimental design, because stimulation of PLC
by FGF could be
demonstrated in 3T3 fibroblasts under similar conditions. This lack of
FGF-induced PLC
phosphorylation and Ca2+ signal in CEC
is in keeping with previous reports examining mitogenic signaling
pathways through FGF in HUVEC (17, 47).
To demonstrate that the increase in intracellular Ca2+
induced by VEGF is causally related to MAPK activation and cell
proliferation, we examined the effect of Ca2+ chelation on
these responses. In our study, treatment of CEC with BAPTA-AM prevented
the rise in intracellular Ca2+ induced by VEGF and
inhibited the activation of MAPK. Because BAPTA-AM did not have an
effect on MAPK activation driven by FGF, it is likely that its activity
on VEGF-driven MAPK phosphorylation is mediated by chelation of
Ca2+ and not by other nonspecific effects. It was reported
previously that mobilization of Ca2+ intracellular stores
in human fibroblasts and A431 cells was sufficient to activate MAPK
(4). Our data suggest that both intracellular
Ca2+ release and flux of external Ca2+ may be
important in endothelial cells, because MAPK inhibition by BAPTA-AM was
observed only at concentrations of the chelator that blocked both the
transient and the sustained Ca2+ signal. Finally, our
results with the PLC inhibitor U-73122 on VEGF-driven cell
proliferation are also consistent with a direct effect on the
PLC
/Ca2+ signaling pathway in VEGF-regulated cell
growth. Again, the lack of effect on FGF-driven responses supports the
hypothesis that PLC
/Ca2+ is not part of the FGF
signaling cascade in endothelial cells. It has been reported, however,
that carboxyamidotriazole (an inhibitor of non-voltage-gated
Ca2+ channels) can inhibit proliferation of HUVEC in
response to bFGF (21). However, Ca2+ responses
were not measured directly in these studies; thus the drug may have had
additional effects other than inhibition of Ca2+ flux.
Our results also raise the question as to what blocks the interaction
of PLC with FGF receptors in endothelial cells. A PLC binding site
(tyr-766) in the kinase domain of the FGF receptor has been identified
(28), giving the receptor the capacity to signal through
PLC
and Ca2+. This has been supported by previous
reports (3, 6, 35, 44) and studies in our own laboratory
demonstrating phosphorylation of PLC
and Ca2+ signal in
nonendothelial cells in response to FGF. The link between FGFR and
PLC
activation in endothelial cells, however, is not clear. Cross et
al. (5) reported an activation of PLC, as measured by
[3H]inositol production, in porcine endothelial cells
overexpressing FGFR, although this pathway does not appear to be linked
to MAPK activation and cell proliferation. The inability to demonstrate similar responses in primary endothelial cells, however, suggests that
something may be unique about the endothelial cell background that
regulates interaction of PLC
with endogenous tyrosine kinase receptors. Such an effect of cell background on signaling pathways was
explored previously by Takahashi and Shibuya (41). In
those studies, PLC
was found to be tyrosine phosphorylated and to
associate with activated VEGFR-2 expressed in both NIH 3T3 cells and
sinusoidal endothelial cells, but rapid activation of MAPK and
subsequent strong mitogenic signaling were generated only in the
endothelial cell background. Similar effects of cell background could
be responsible for the differences we observe between FGF-driven PLC
phosphorylation and Ca2+ responses in endothelial cells vs.
3T3 fibroblasts.
Recently, the Src homology domain 2 (SH2) adapter protein VRAP has been
identified as a protein that binds the intracellular domain of VEGFR-2
and constitutively associates with PLC (47). These
studies suggested that VRAP recruits PLC
to the activated VEGF
receptor and regulates the interaction of the receptor with effector
proteins. VRAP binding to other RTK has not yet been determined. If
PLC
bound to VRAP is selectively recruited to VEGF-activated
VEGFR-2, this could minimize accessibility of PLC
to FGFR and other
tyrosine kinase receptors. Such an idea was recently explored in
studies of the docking protein FRS2 (32). It was shown
that FRS2 binds to FGFR-1 and nerve growth factor receptor TrkA in a
phosphorylation-independent and -dependent manner, respectively, and
that FRS2 interaction with TrkA is diminished in cells overexpressing
FGFR-1. The authors suggested that interacting pathways might regulate
receptor signaling by sequestering a common element, which both
receptors use to transmit their signals. This phenomenon could explain
our inability to demonstrate PLC
phosphorylation and
Ca2+ signaling through FGFR in endothelial cells and is
currently under investigation.
In summary, our examination of signaling by VEGF and FGF in CEC
indicates differences in the pathways used by these receptors in
promoting mitogenesis. MAPK activation is coupled to both VEGF- and
FGF-stimulated growth, as previously reported in HUVEC. PLC and
Ca2+, however, are shown to play a direct role in VEGF- but
not FGF-mediated MAPK activation and cell proliferation. These
observations represent an example of tissue-specific regulation of RTK
signaling. Elucidation of signaling mechanisms triggering the
functional effects of different growth factors will be important in
understanding regulation of angiogenesis and in the development of
novel therapies for neovasculature-based diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Dr. G. Rodrigues for helpful discussions during the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: G. W. De Vries, Dept. of Biological Sciences, Allergan, Inc., 2525 Dupont Drive, Irvine, CA 92612 (E-mail: devries_gerald{at}allergan.com).
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.
Received 17 April 2001; accepted in final form 21 June 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alessi, DR,
Cuenda A,
Cohen P,
Dudley DT,
and
Saltiel AR.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem
270:
27489-27494,
1995
2.
Brock, TA,
Dvorak HF,
and
Senger DR.
Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells.
Am J Pathol
138:
213-221,
1991[Abstract].
3.
Burgess, WH,
Dionne CA,
Kaplow J,
Mudd R,
Friesel R,
Zilberstein A,
Schlessinger J,
and
Jaye M.
Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.
Mol Cell Biol
10:
4770-4777,
1990[ISI][Medline].
4.
Chao, TS,
Byron KL,
Lee K-M,
Villereal M,
and
Rosner MR.
Activation of MAP kinases by calcium-dependent and calcium-independent pathways.
J Biol Chem
267:
19876-19883,
1992
5.
Cross, MJ,
Hodgkin MN,
Roberts S,
Landgren E,
Wakelam MJO,
and
Claesson-Welsh L.
Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGF-stimulation of phospholipase C, phospholipase D, phospholipase A2, phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells.
J Cell Sci
113:
643-651,
2000
6.
Cuadrado, A,
and
Molloy CJ.
Overexpression of phospholipase C- in NIH 3T3 fibroblasts results in increased phosphatidylinositol hydrolysis in response to platelet-derived growth factor and basic fibroblast growth factor.
Mol Cell Biol
10:
6069-6072,
1990[ISI][Medline].
7.
Cunningham, SA,
Arrate MP,
Brock TA,
and
Waxham MN.
Interactions of FLT-1 and KDR with phospholipase C : identification of the phosphotyrosine binding sites.
Biochem Biophys Res Commun
240:
635-639,
1997[ISI][Medline].
8.
Cunningham, SA,
Tran TM,
Arrate MP,
Bjercke R,
and
Brock TA.
KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells.
Am J Physiol Cell Physiol
276:
C176-C181,
1999
9.
D'Angelo, G,
Struman I,
Martial J,
and
Weiner RI.
Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.
Proc Natl Acad Sci USA
92:
6374-6378,
1995[Abstract].
10.
Doans, AM,
Hegland DD,
Sethi R,
Kovesdi I,
Bruder JT,
and
Finkel T.
VEGF stimulates MAPK through a pathway that is unique for receptor tyrosine kinases.
Biochem Biophys Res Commun
255:
545-548,
1999[ISI][Medline].
11.
Dougher, M,
and
Terman BI.
Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization.
Oncogene
18:
1619-1627,
1999[ISI][Medline].
12.
Guo, D,
Jia Q,
Song H-Y,
Warren RS,
and
Donner DB.
Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains.
J Biol Chem
270:
6729-6733,
1995
13.
Guo, D-Q,
Wu L-W,
Dunbar JD,
Ozes ON,
Mayo LD,
Kessler KM,
Gustin JA,
Baerwald MR,
Jaffe EA,
Warren RS,
and
Donner DB.
Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.
J Biol Chem
275:
11216-11221,
2000
14.
Hubbard, SR.
Structural analysis of receptor tyrosine kinases.
Prog Biophys Mol Biol
71:
343-358,
1999[ISI][Medline].
15.
Hughes, SE.
Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues.
J Histochem Cytochem
45:
1005-1019,
1997
16.
Kendall, RL,
Rutledge RZ,
Mao X,
Tebben AJ,
Hungate RW,
and
Thomas KA.
Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues.
J Biol Chem
274:
6453-6460,
1999
17.
Kent, KC,
Mii S,
Harrington EO,
Chang JD,
Mallette S,
and
Ware JA.
Requirement for protein kinase C activation in basic fibroblast growth factor-induced human endothelial cell proliferation.
Circ Res
77:
231-238,
1995
18.
Kim, HK,
Kim JW,
Zilberstein A,
Margolis B,
Kim JG,
Schlessinger J,
and
Rhee SG.
PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254.
Cell
65:
435-441,
1991[ISI][Medline].
19.
Kimura, H,
Spee C,
Sakamoto T,
Hinton DR,
Ogura Y,
Tabata Y,
Ikada Y,
and
Ryan SJ.
Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres.
Invest Ophthalmol Vis Sci
40:
524-528,
1999[Abstract].
20.
Klint, P,
Kanda S,
and
Claesson-Welsh L.
Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells.
J Biol Chem
270:
23337-23344,
1995
21.
Kohn, EC,
Alessandro R,
Spoonster J,
Wersto RP,
and
Liotta LA.
Angiogenesis: role of calcium-mediated signal transduction.
Proc Natl Acad Sci USA
92:
1307-1311,
1995[Abstract].
22.
Kouhara, H,
Hadari YR,
Spivak-Kroizman T,
Schilling J,
Bar-Sagi D,
Lax I,
and
Schlessinger J.
A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway.
Cell
89:
693-702,
1997[ISI][Medline].
23.
Kwak, N,
Okamota N,
Wood JM,
and
Campochiaro PA.
VEGF is major stimulator in model of choroidal neovascularization.
Invest Ophthalmol Vis Sci
41:
3158-3164,
2000
24.
Larrivee, B,
and
Karsan A.
Signaling pathways induced by vascular endothelial growth factor.
Int J Mol Med
5:
447-456,
2000[ISI][Medline].
25.
La Vallee, TM,
Prudovsky IA,
McMahon GA,
Hu X,
and
Maciag T.
Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the src pathway correlates with migration.
J Cell Biol
141:
1647-1658,
1998
26.
Mandriota, SJ,
and
Pepper MS.
Vascular endothelial growth factor-induced in vitro angiogenesis and plasminogen activator expression are dependent on endogenous basic fibroblast growth factor.
J Cell Sci
110:
2293-2302,
1997
27.
Matsushima, M,
Ogata N,
Takada Y,
Tobe T,
Yamada H,
Takahashi K,
and
Uyama M.
FGF receptor 1 expression in experimental choroidal neovascularization.
Jpn J Ophthalmol
40:
329-338,
1996[ISI][Medline].
28.
Mohammadi, M,
Honegger AM,
Rotin D,
Fischer R,
Bellot F,
Li W,
Dionne CA,
Jaye M,
Rubinstein M,
and
Schlessinger J.
A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (flg) is a binding site for the SH2 domain of phospholipase C-1.
Mol Cell Biol
11:
5068-5078,
1991[ISI][Medline].
29.
Morales-Ruiz, M,
Fulton D,
Sowa G,
Languino LR,
Fujio Y,
Walsh K,
and
Sessa WC.
Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt.
Circ Res
86:
892-896,
2000
30.
Morse, LS,
Terrell J,
and
Sidikaro Y.
Bovine retinal pigment epithelium promotes proliferation of choroidal endothelium in vitro.
Arch Ophthalmol
107:
1659-1663,
1989[Abstract].
31.
Nishibe, S,
Wahl MI,
Hernandez-Sotomayor SM,
Tonks NK,
Rhee SG,
and
Carpenter G.
Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation.
Science
250:
1253-1256,
1990[ISI][Medline].
32.
Ong, SH,
Guy GR,
Hadari YR,
Laks S,
Gotoh N,
Schlessinger J,
and
Lax I.
FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.
Mol Cell Biol
20:
979-989,
2000
33.
Pepper, MS,
Mandriota SJ,
Jeltsch M,
Kumar V,
and
Alitalo K.
Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity.
J Cell Physiol
177:
439-452,
1998[ISI][Medline].
34.
Pepper, MS,
Mandriota SJ,
Vassalli J-D,
Orci L,
and
Montesano R.
Angiogenesis-regulating cytokines: activities and interactions.
Curr Top Microbiol Immunol
213:
31-67,
1996[ISI][Medline].
35.
Peters, KG,
Marie J,
Wilson E,
Ives HE,
Escobedo J,
Rosario MD,
Mirda D,
and
Williams LT.
Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca 2+ flux but not mitogenesis.
Nature
358:
678-684,
1992[ISI][Medline].
36.
Petrova, TV,
Makinen T,
and
Alitalo K.
Signaling via vascular endothelial growth factor receptors.
Exp Cell Res
253:
117-130,
1999[ISI][Medline].
37.
Risau, W.
Mechanisms of angiogenesis.
Nature
386:
671-674,
1997[ISI][Medline].
38.
Schlessinger, J,
and
Ullrich A.
Growth factor signaling by receptor tyrosine kinases.
Neuron
9:
383-391,
1992[ISI][Medline].
39.
Smith, RJ,
Sam LM,
Justen JM,
Bundy GL,
Bala GA,
and
Bleasdale JE.
Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.
J Pharmacol Exp Ther
253:
688-697,
1990[Abstract].
40.
Szebenyi, G,
and
Fallon JF.
Fibroblast growth factors as multifunctional signaling factors.
Int Rev Cytol
185:
45-106,
1999[ISI][Medline].
41.
Takahashi, T,
and
Shibuya M.
The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC- pathway and partially induces mitotic signals in NIH3T3 fibroblasts.
Oncogene
14:
2079-2089,
1997[ISI][Medline].
42.
Takahashi, T,
Ueno H,
and
Shibuya M.
VEGF activates protein kinase C-dependent, but ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells.
Oncogene
18:
2221-2230,
1999[ISI][Medline].
43.
Thakker, GD,
Hajjar DP,
Muller WA,
and
Rosengart TK.
The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling.
J Biol Chem
274:
10002-10007,
1999
44.
Tsuda, T,
Kaibuchi K,
Kawahara Y,
Fukuzaki H,
and
Takai Y.
Induction of protein kinase C activation and Ca2+ mobilization by fibroblast growth factor in Swiss 3T3 cells.
FEBS Lett
191:
205-210,
1985[ISI][Medline].
45.
Wada, M,
Ogata N,
Otsuji T,
and
Uyama M.
Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization.
Curr Eye Res
18:
203-213,
1999[ISI][Medline].
46.
Wellner, M,
Maasch C,
Kupprion C,
Lindschau C,
Luft FC,
and
Haller H.
The proliferative effect of vascular endothelial growth factor requires protein kinase C- and protein kinase C-
.
Arterioscler Thromb Vasc Biol
19:
178-185,
1999
47.
Wu, L-W,
Mayo LD,
Dunbar JD,
Kessler KM,
Baerwald MR,
Jaffe EA,
Wang D,
Warren RS,
and
Donner DB.
Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.
J Biol Chem
275:
5096-5103,
2000
48.
Wu, L-W,
Mayo LD,
Dunbar JD,
Kessler KM,
Ozes ON,
Warren RS,
and
Donner DB.
VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor.
J Biol Chem
275:
6059-6062,
2000
49.
Xia, P,
Aiello LP,
Ishii H,
Jiang ZY,
Park DJ,
Robinson GS,
Takagi H,
Newsome WP,
Jirousek MR,
and
King GL.
Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.
J Clin Invest
98:
2018-2026,
1996
50.
Yu, Y,
and
Sato JD.
MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor.
J Cell Physiol
178:
235-246,
1999[ISI][Medline].
51.
Zachary, I.
Vascular endothelial growth factor: how it transmits its signal.
Exp Nephrol
6:
480-487,
1998[ISI][Medline].