Role of PLCgamma and Ca2+ in VEGF- and FGF-induced choroidal endothelial cell proliferation

A. P. McLaughlin and G. W. De Vries

Department of Biological Sciences, Allergan, Incorporated, Irvine, California 92612


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)gamma and Ca2+ in their downstream signaling cascades are still not clear. We have examined the effects of VEGF and FGF on PLCgamma phosphorylation and on changes in intracellular Ca2+ levels in primary endothelial cells. VEGF stimulation leads to PLCgamma 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 PLCgamma 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 PLCgamma 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)gamma (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 PLCgamma (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 PLCgamma 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 PLCgamma 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 PLCgamma 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 PLCgamma 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 PLCgamma 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 PLCgamma 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 PLCgamma and Ca2+ in regulating cell growth by directly comparing VEGF- and FGF-stimulated primary endothelial cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PLCgamma 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, PLCgamma 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 PLCgamma 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). PLCgamma 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptor autophosphorylation in primary cultures of choroidal endothelial cells (CEC). A: total cell lysates prepared from untreated (-) or VEGF-stimulated (+) (10 ng/ml for 5 min) cells were resolved by SDS-PAGE and immunoblotted (IB) with anti-pTyr (left) or anti-VEGF receptor (VEGFR)-2 (right) antibodies. B: lysates prepared from untreated (-) or FGF-stimulated (+; 10 ng/ml + heparin 10 µg/ml for 5 min) cells were immunoprecipitated with an antiserum recognizing FGF receptor (FGFR)-1. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with anti-pTyr (left) or anti-FGFR-1 (right). Position of the 116-kDa phosphorylated form of FGFR-1 detected is indicated by the arrow on the right, and the position of the corresponding molecular mass marker is indicated on the left.

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.


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Fig. 2.   Time course of VEGF- and FGF-dependent activation of mitogen-activated protein (MAP) kinase (MAPK) and inhibition by PD-98059. Quiescent CEC were stimulated with 10 ng/ml VEGF or basic FGF (bFGF) for the indicated times (A) or pretreated with vehicle or increasing concentrations of PD-98059 (30 min) before growth factor stimulation for 5 min (B). Cell lysates prepared as described in MATERIALS AND METHODS were analyzed by SDS-PAGE and blotted (IB) with anti-phospho-specific MAPK antibodies (p-MAPK) or anti-MAPK antibodies (MAPK) to quantitate total MAPK protein levels in samples. The arrows indicate the position of p44 and p42 MAPK.

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.


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Fig. 3.   VEGF- and bFGF-stimulated proliferation and inhibition by PD-98059. Cells were stimulated with mitogen in the presence or absence of inhibitor for 48 h in medium containing 0.5% FBS. Cell proliferation was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA using enzyme immunoassay. A: VEGF and bFGF growth curves in CEC. Cells were left untreated or cultured in the presence of increasing concentrations of VEGF or bFGF as indicated. Results are expressed as % of control cells cultured in the absence of growth factor. B: inhibition of growth in (A) by PD-98059. Cells were incubated in absence and presence of increasing concentrations of inhibitor for 30 min followed by mitogen stimulation (10 ng/ml) and 48-h growth. Results are expressed as % inhibition of growth factor-stimulated response in absence of inhibitor. All values shown are means ± SE from 3 independent experiments. *P < 0.05.

Role of PLCgamma /Ca2+ pathway in VEGF- and bFGF-mediated proliferation. PLCgamma 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).


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Fig. 4.   VEGF but not bFGF induces intracellular Ca2+ signaling in CEC. Cells (1 × 106) were loaded with fura 2-AM to monitor intracellular Ca2+ concentration in untreated cells (baseline) or cells treated with 10 ng/ml VEGF or bFGF (added at arrow).

Increases in intracellular Ca2+ have been correlated with activation of PLCgamma . Because increased enzyme activity has been associated with tyrosine phosphorylation (18, 31), we examined the ability of both VEGF and FGF to stimulate PLCgamma tyrosine phosphorylation in CEC. PLCgamma was immunoprecipitated from control and VEGF-stimulated cells. Western blot analysis identified phosphorylated PLCgamma only in lysates from VEGF-stimulated cells (Fig. 5A). Protein levels were verified by blotting PLCgamma immunoprecipitates with anti-PLCgamma antibodies. In addition, a 230-kDa phosphorylated protein consistent with the molecular mass of VEGFR-2 was coimmunoprecipitated with phosphorylated PLCgamma from VEGF-stimulated lysates. Reblotting with anti-flk-1 antibody identified this protein as VEGFR-2 (data not shown). These data confirm VEGF-stimulated PLCgamma phosphorylation and association with activated endogenous VEGF receptors in CEC. In contrast to VEGF, bFGF did not induce PLCgamma phosphorylation in CEC (Fig. 5B). Altering length of stimulation and bFGF concentration did not affect this result. As a positive control, we verified FGF-stimulated PLCgamma phosphorylation in 3T3 fibroblasts under the same experimental conditions (data not shown). The differential effects of VEGF and FGF on PLCgamma phosphorylation are consistent with their respective effects on intracellular Ca2+ levels. These data support a role for PLCgamma /Ca2+ in VEGF, but not FGF, RTK signal transduction in CEC.


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Fig. 5.   VEGF but not bFGF induces phospholipase C (PLC)gamma phosphorylation in CEC. Lysates prepared from untreated (-) and 10 ng/ml VEGF-stimulated (A) or 10 ng/ml bFGF-stimulated (B) cells (+) were immunoprecipitated with an antibody to PLCgamma . Immunocomplexes were resolved by SDS-PAGE and immunoblotted (IB) with anti-pTyr (left panels) or anti-PLCgamma (right panels) to show equal loading of protein. VEGFR-2 coimmunoprecipitates with PLCgamma in VEGF-stimulated cells as indicated by the arrows in A. VEGF was used as a positive control in B, confirming PLCgamma phosphorylation in response to VEGF but not FGF in endothelial cells.

Role of Ca2+ in VEGF- and bFGF-induced activation of MAPK. To determine whether PLCgamma /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 PLCgamma and Ca2+ in the signaling mediating proliferation through VEGF but not FGF receptors in CEC.


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Fig. 6.   1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM inhibits VEGF Ca2+ signaling (A) and MAPK activation (B) in CEC. A: VEGF Ca2+ response and complete inhibition in the presence of 25 µM BAPTA-AM (left). VEGF Ca2+ signaling was monitored in the presence and absence of increasing concentrations of BAPTA-AM. Increases in sustained or transient intracellular Ca2+ levels ([Ca2+]i) were determined after 30-min preincubation with BAPTA-AM followed by VEGF stimulation (10 ng/ml; right). Data are representative of 2 independent experiments. B: quiescent CEC were pretreated with vehicle or increasing concentrations of BAPTA (30 min) before VEGF (left) or bFGF (right) stimulation for 5 min. Cell lysates prepared as described in MATERIALS AND METHODS were analyzed by SDS-PAGE and blotted (IB) with anti-phospho-specific MAPK antibodies. Data are representative of 3 independent experiments.

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 PLCgamma in the VEGF-induced mitogenic response in endothelial cells.


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Fig. 7.   Effect of PLCgamma inhibitor U-73122 on VEGF- and bFGF-mediated growth. Cells were incubated in the absence and presence of increasing concentrations of inhibitor for 30 min followed by mitogen stimulation (10 ng/ml) and 48-h growth. Cell proliferation was determined by BrdU incorporation into DNA using enzyme immunoassay. Results are expressed as % inhibition of growth factor-stimulated response in absence of inhibitor. All values shown are means ± SE from 3 independent experiments. *P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PLCgamma 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 PLCgamma has been inferred from previous studies. Xia et al. (49) reported that in porcine aortic endothelial cells, VEGF stimulation led to PLCgamma 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 PLCgamma , which is correlated with MAPK activation and increased 3H-labeled thymidine incorporation into DNA (47). It has been argued, in fact, that the PLCgamma /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 PLCgamma activity. However, in cells expressing a mutant receptor that fails to stimulate PLCgamma , 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 PLCgamma 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 PLCgamma phosphorylation by VEGF but not FGF. It is unlikely that the lack of response in CEC is due to experimental design, because stimulation of PLCgamma by FGF could be demonstrated in 3T3 fibroblasts under similar conditions. This lack of FGF-induced PLCgamma 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 PLCgamma inhibitor U-73122 on VEGF-driven cell proliferation are also consistent with a direct effect on the PLCgamma /Ca2+ signaling pathway in VEGF-regulated cell growth. Again, the lack of effect on FGF-driven responses supports the hypothesis that PLCgamma /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 PLCgamma 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 PLCgamma and Ca2+. This has been supported by previous reports (3, 6, 35, 44) and studies in our own laboratory demonstrating phosphorylation of PLCgamma and Ca2+ signal in nonendothelial cells in response to FGF. The link between FGFR and PLCgamma 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 PLCgamma 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, PLCgamma 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 PLCgamma 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 PLCgamma (47). These studies suggested that VRAP recruits PLCgamma 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 PLCgamma bound to VRAP is selectively recruited to VEGF-activated VEGFR-2, this could minimize accessibility of PLCgamma 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 PLCgamma 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. PLCgamma 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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