©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Cytosolic Phospholipase A by Basic Fibroblast Growth Factor via a p42 Mitogen-activated Protein Kinase-dependent Phosphorylation Pathway in Endothelial Cells (*)

(Received for publication, July 20, 1994; and in revised form, November 15, 1994 )

Gaurisankar Sa (1)(§) Gurunathan Murugesan (1)(§) Michael Jaye (2) Yuri Ivashchenko (2) Paul L. Fox (1)(¶)

From the  (1)Department of Cell Biology, Cleveland Clinic Research Institute, Cleveland, Ohio 44195 and the (2)Molecular Biology Division, Rhône-Poulenc-Rorer Central Research, Collegeville, Pennsylvania 19426

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2) (PLA(2))-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(2) (cPLA(2)) by demonstrating in EC lysates a requirement for micromolar Ca, dithiothreitol insensitivity, and inactivation by anti-cPLA(2) antiserum. The role of cPLA(2) is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA(2) phosphorylation, and cPLA(2) translocation from cytosol to membranes. Phosphorylation of cPLA(2), 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(2) may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.


INTRODUCTION

Basic fibroblast growth factor (FGF) (^1)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-C1(3) , a 34-kDa lipocortin-like protein(4) , cortactin(5) , and Shc(6) . Of these proteins only phospholipase-C1 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(2) (PLA(2)) inhibitors, albeit of uncertain specificity, almost completely block basic FGF-stimulated arachidonate release(10, 12) . Direct measurement of PLA(2) activity by an ``in vitro'' assay of EC lysates using L-alpha-1-palmitoyl-2-[1-^14C]arachidonyl phosphatidylcholine (PC) as substrate has shown that basic FGF stimulates PLA(2) activity by about 80%(10) .

At least two distinct forms of PLA(2) 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(2)) is homologous to forms secreted by the pancreas. A second form, cytosolic PLA(2) (cPLA(2)), has been recently identified and purified from human monocytic cells (14) and cloned from mRNA isolated from U937 cells(15, 16) . Cytosolic PLA(2) 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(2) 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(2)(15) .

In this report we show that the PLA(2) activated by basic FGF is cPLA(2), 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.


EXPERIMENTAL PROCEDURES

Reagents

Human recombinant basic FGF was obtained from Austral Biologicals and Upstate Biotechnology. Sucrose monolaurate (SM-1200) was a gift from the Mitsubishi-Kasei Co. (Tokyo, Japan). Leupeptin, antipain, pepstatin A, and calf intestinal phosphatase were purchased from Boehringer Mannheim, bovine thrombin was from Pentex-Miles, and all solvents were from Fisher. Dithiothreitol, benzamidine, bacitracin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride (PMSF), phorbol 12-myristate 13-acetate (PMA), and all other reagents were from Sigma. Rabbit anti-human cPLA(2) antiserum was prepared by the solid-phase multiple antigenic peptide method(21) . The peptide TPDSRKRTRHFNNDINPVWN (residues 53-72 of human cPLA(2)(15) ) was synthesized on Fmoc (N-(9-fluorenyl)methoxycarbonyl) multiple antigen peptide resin, 8-branch (Applied Biosystems), cleaved from the resin with trifluoroacetate, purified by ion exchange chromatography, and analyzed by reverse phase high performance liquid chromatography, SDS-PAGE, and ion spray mass spectrometry. A rabbit was injected with 1 mg of the antigen in complete Freund's adjuvant and then six times with the antigen in incomplete adjuvant. The antibody tested positive by immunoblot analysis of human recombinant cPLA(2) produced in Kluveromyces lactis(^2)Immunoblot analysis of a lysate from U937 cells revealed two approximately 85-kDa bands of cPLA(2) (representing the unphosphorylated and phosphorylated forms) with no other bands detected. Rabbit polyclonal anti-p42 MAP kinase was generously provided by Dr. Michael Weber(8) .

Preparation of EC Membrane and Cytosolic Fractions

EC were isolated from bovine aorta and maintained in Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1, v/v) containing 5% fetal bovine serum(22) . EC were grown to 70-80% confluence and transferred to serum-free medium containing 1 mg/ml gelatin for at least 24 h before use. The cells were scraped and homogenized in 10 mM Hepes buffer (pH 7.5) containing 250 mM sucrose, 5 mM EGTA, and protease inhibitors (bacitracin, 3 µg/ml; benzamidine, 100 µg/ml; leupeptin, 2 µg/ml; pepstatin A, 2 µg/ml; trypsin inhibitor, 2 µg/ml; PMSF, 2 µg/ml; and antipain, 20 µg/ml). The homogenate was centrifuged at 900 times g for 5 min to remove cell debris and intact cells. The supernatant was centrifuged at 13,000 times g for 1 h, and the pellets were harvested and washed with 20 mM Hepes buffer (pH 7.5) containing 10 mM MgCl(2), 5 mM EGTA, and the protease inhibitors listed above. EC membranes were purified by Percoll gradient centrifugation (23) and suspended at 10 mg of protein/ml. To obtain the cytosolic fraction, the supernatants from the ``membrane'' centrifugation were recentrifuged at 50,000 times g for 1 h, and the supernatants adjusted to 10 mg of protein/ml in buffer containing 10% glycerol and stored at -80 °C.

Determination of in Vitro Phospholipase A(2) Activity

Phospholipase A(2) activity in EC lysates and cellular fractions was determined by an in vitro assay by a modification of previous methods(24) . In brief, L-alpha-1-palmitoyl-2-[^14C]arachidonyl PC (53 mCi/mmol, DuPont NEN) was dried under N(2), resuspended in dimethyl sulfoxide by vigorous mixing for 2 min, and dispersed in a bath sonicator. The substrate (5 µl containing 1.5 times 10^5 cpm) was incubated with EC fractions at 37 °C for 30 min in a total volume of 0.2 ml of 25 mM Hepes (pH 7.5), containing 200 mM sucrose, protease inhibitors (10 µg/ml benzamidine and trypsin inhibitor, 20 µg/ml bacitracin and PMSF), phosphatase inhibitors (5 µM of o-phosphoserine, o-phosphotyrosine, o-phosphothreonine, beta-glycerophosphate, p-nitrophenyl phosphate, and sodium vanadate), and 0.05% sucrose monolaurate in the presence of 900 µM Ca and 5 mM dithiothreitol. The reaction was stopped with 0.1 ml of ice-cold ethanol containing 2% (v/v) glacial acetic acid and 100 µg/ml of unlabeled arachidonate. Heptane (1.5 ml) and water (1.0 ml) were added and mixed. The organic phase was dried and dissolved in chloroform, and radioactivity in the arachidonate band was determined after thin layer chromatography.

Cellular Release of [^3H]Arachidonate

Arachidonate release was measured from prelabeled EC essentially as described(25) . Confluent EC monolayers were incubated with 0.2-0.5 µCi/ml of [^3H]arachidonate (100 Ci/mmol, DuPont NEN) for 24 h at 37 °C. The cells were then incubated for 15-30 min with serum-free medium containing agonists plus 0.3% fatty acid-free bovine serum albumin to trap released fatty acids. To measure [^3H]arachidonate release, the cells were placed on ice, the conditioned medium collected, and radioactivity determined in a liquid scintillation counter.

Immunoprecipitation of p42 MAP Kinase

Quiescent, semi-confluent EC were prelabeled for 3 h with [P]orthophosphate (300 µCi/ml) in phosphate-free media and then stimulated with basic FGF (10 ng/ml) for up to 20 min. The cells were washed with ice-cold phosphate-buffered saline (PBS) and the reaction stopped by immersion in a mixture of dry ice and ethanol. The cells were lysed by sonication in buffer containing the protease and phosphatase inhibitor mixture described above. To remove p42 MAP kinase, the lysate was incubated with rabbit polyclonal anti-p42 MAP kinase antibody for 4 h at 4 °C and then with protein A-Sepharose beads (precoated with 3% non-fat dry milk for 1 h and washed by centrifugation)(26) . The immunoprecipitate was centrifuged at 12,000 rpm for 2 min at 4 °C and the pellet washed with ice-cold PBS containing phosphatase inhibitors. The pellets were suspended in Laemmli buffer, boiled for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatants were subjected to SDS-PAGE (10% acrylamide) and autoradiography.

Immunoblot Analysis of cPLA(2) and Mobility Shift Assay of Phosphorylation

Semi-confluent bovine aortic EC in 150-mm dishes were used for immunoblot analysis. The medium was aspirated, the monolayer washed with ice-cold PBS, and the dishes cooled in a dry ice/ethanol slurry. The cells were lysed by sonication in buffer containing protease and phosphatase inhibitors. The samples were treated with 2 times Laemmli buffer, boiled for 5 min and subjected to 7.5% SDS-PAGE (20 mA/gel) using acrylamide:bisacrylamide (60:1). For detection of cPLA(2), electrophoresis was continued for 3 h after the tracking dye exited the gel to increase separation between the native and phosphorylated isoforms. The samples were transferred to Immobilon-P membranes (Millipore) at 2.5 mA/cm^2 for 45-60 min. Blocking was done for 2 h at 45 °C in PBS containing 5% non-fat dry milk, 1% bovine calf serum, and 0.1% Tween 20. The blots were incubated for 2 h with rabbit anti-human cPLA(2) (1:5000) and then with horseradish peroxidase-conjugated goat anti-rabbit IgG antisera (1:5000) in buffer containing 0.1% Tween 20, 3% phosphatase- and peroxidase-free bovine serum albumin, and 1% bovine calf serum. The signal was detected by chemiluminescence (Amersham Corp.) and quantitated by densitometry. The amounts of cell extracts applied to gels were normalized by protein concentration measured by a Bio-Rad DC assay using bovine serum albumin as a standard.

Measurement of EC Migration

To measure cell migration, confluent EC cultures were wounded with a blade drawn through the monolayer to remove cells on one side of the cut(27) . The medium was replaced with Dulbecco's modified Eagle's medium containing 1 mg/ml of gelatin and cell movement quantitated after 22 h as described(27) .


RESULTS

Effect of Basic FGF on Phospholipase A(2) Activity in EC Fractions

We showed previously that lysates prepared from basic FGF-stimulated EC had elevated PLA(2) activity as measured by arachidonate release from L-alpha-1-palmitoyl-2-[1-^14C]arachidonyl PC (10) . To determine the intracellular location of the phospholipase, lysates from unstimulated EC were fractionated into membrane and cytosolic fractions and in vitro PLA(2) activity was determined. PLA(2) activity in EC membranes was low, but was stimulated by basic FGF by about 45% (Fig. 1A). The activity in the cytosolic fraction was substantially higher than in membranes, but as expected in the absence of membrane receptors, it was not stimulated by basic FGF. When the cytosol and membrane fractions were combined, the stimulation of PLA(2) activity was clearly synergistic (Fig. 1A). A mechanism consistent with these observations is that the PLA(2) activity present in membranes is due to cPLA(2) and that the amount of activity is lower than that in the cytosol due to limited translocation in the absence of agonist. However, when the cPLA(2) activity in the cytosolic fraction is added to membranes, basic FGF stimulates translocation and activation of the enzyme. Since other mechanisms could also explain these results, e.g. stimulation of a non-cPLA(2) by a cytosolic activating protein, translocation of the activity was examined directly by comparing fractions from cells pretreated with or without basic FGF. This experiment shows that the increase in PLA(2) activity in membranes from basic FGF-pretreated cells is accompanied by decreased activity in the cytosolic fraction, a finding consistent with translocation of cPLA(2) (Fig. 1B).


Figure 1: Phospholipase A(2) 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(2) activity was measured by hydrolysis of L-alpha-1-palmitoyl-2-[^14C]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.



Basic FGF Stimulates Cytosolic PLA(2) Activity

The activity of cPLA(2) was shown previously to be maximal in the presence of micromolar Ca, whereas the secretory form of the enzyme required millimolar Ca(13) . The Ca requirement of the basic FGF-stimulated PLA(2) present in EC was thus measured in reconstituted EC lysates. The cytosolic fraction of EC lysates was preincubated with Chelex 100 resin (Bio-Rad) to remove Ca and then reconstituted with the membrane fraction in the presence of exogenous Ca. When measured as release of arachidonate from L-alpha-1-palmitoyl-2-[1-^14C]arachidonyl PC, unstimulated cells exhibited a biphasic activation of PLA(2) by Ca (Fig. 2). Parameter estimation by nonlinear regression analysis using the Michaelis-Menten equation (28) indicated that 35% of the unstimulated activity (i.e. in the absence of basic FGF) at 10 mM Ca was due to an enzyme with half-maximal activity at about 0.3 µM Ca and near-maximal activity at 10 µM Ca. The enzyme responsible for the remaining unstimulated activity had half-maximal activity at about 1 mM Ca and near-maximal activity at a concentration higher than 10 mM. The unstimulated activity observed was likely due in part to the presence of endogenous basic FGF, since the activity was blocked by anti-basic FGF antibody (not shown). Addition of basic FGF to the reconstituted lysates caused a substantial increase in PLA(2) activity at all Ca concentrations above 10 nM. Basic FGF stimulated PLA(2) activity by 2-fold at 10 µM Ca, a concentration at which essentially all activity is due to cPLA(2). When specific growth factor-mediated PLA(2) activity was calculated by difference, it was apparent that basic FGF stimulated a single activity that required low Ca concentrations; the half-maximal activity was estimated to be about 0.1 µM Ca (Fig. 2). These data are consistent with the presence of two distinct PLA(2) enzymes, differing in Ca sensitivity, and stimulation by basic FGF of the form with a low Ca requirement. In view of results showing that cPLA(2) purified from human monocytic cells has a bimodal Ca requirement, a second mechanism is also feasible(14) , namely, that cPLA(2) is the only PLA(2) enzyme present and that basic FGF stimulates only that activity of cPLA(2) that requires low Ca.


Figure 2: Ca dependence of basic FGF-stimulated PLA(2) 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 (bullet) or absence (circle) of 10 ng/ml basic FGF for 15 min. PLA(2) activity in the mixture was determined by hydrolysis of L-alpha-1-palmitoyl-2-[^14C]arachidonyl PC in the presence of various concentrations of exogenous Ca. The difference is the activity due to basic FGF (times). 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(2) on the activity of EC lysates was tested using the in vitro PLA(2) assay. The antibody blocked essentially all basic FGF-stimulated activity, confirming that the PLA(2) activated by basic FGF in EC is cPLA(2) (Fig. 3). The activity of purified cPLA(2) is insensitive to reduction by dithiothreitol(14) ; likewise, the basic FGF-stimulated PLA(2) activity in EC lysates was found to be insensitive to dithiothreitol (<3% inhibition, not shown) providing further confirmation of the role of cPLA(2).


Figure 3: Inhibition of basic FGF-stimulated arachidonate release by anti-cPLA(2). An EC lysate was incubated in the presence of rabbit anti-cPLA(2) antiserum (or control serum) for 1 h at 4 °C before assay. Phospholipase A(2) activity was measured as the release of [^14C]arachidonate in the presence (solid bars) or absence (striped bars) of 10 ng/ml basic FGF.



Requirement for p42 MAP Kinase for Activation of cPLA(2) by Basic FGF

Phosphorylation of cPLA(2) by p42 MAP kinase is required for maximal activity of the enzyme in vitro(18) . EC lysates were therefore depleted of p42 MAP kinase by repeated immunoprecipitation and PLA(2) activity was measured as release of [1-^14C]arachidonate from its PC precursor. Essentially all basic FGF-stimulated PLA(2) activity was blocked by this treatment, demonstrating the critical regulatory role of MAP kinase (Fig. 4). Treatment of lysates with calf intestinal phosphatase also was also inhibitory, confirming the specific role of phosphorylation. Basic FGF stimulated the tyrosine phosphorylation of p42 MAP kinase (Fig. 5A); maximal activation was nearly 3-fold at 10 min and returned almost to base line by 20 min as reported in fibroblasts(8) . Basic FGF also increased tyrosine phosphorylation of p42 MAP kinase, as measured by immunoprecipitation with anti-p42 MAP kinase followed by immunoblotting with anti-phosphotyrosine, and by kinase activity using myelin basic protein as substrate (data not shown).


Figure 4: Role of p42 MAP kinase-mediated phosphorylation in basic FGF-stimulated PLA(2) 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(2) activity of the combined fractions was determined by hydrolysis of L-alpha-1-palmitoyl-2-[^14C]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(2) activity.




Figure 5: Time course of basic FGF-stimulated phosphorylation of p42 MAP kinase and cPLA(2) in EC. A, semi-confluent EC cultures (25 times 10^6 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 times 10^6 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(2). C, phosphorylation of cPLA(2) (box) and p42 MAP kinase () were quantitated by densitometry and expressed as the percent of total immunodetectable cPLA(2) and relative densitometric units, respectively.



Basic FGF Stimulates Phosphorylation and Translocation of cPLA(2)

Activation of cPLA(2) by growth factors is thought to require phosphorylation reactions involving MAP kinase or protein kinase C or both(18, 19, 20, 29) . The decreased electrophoretic mobility or ``mobility shift'' of the phosphorylated protein during SDS-PAGE (18) was used to investigate phosphorylation of cPLA(2) by basic FGF. Semi-confluent aortic EC were treated with 10 ng/ml of basic FGF for up to 30 min and lysates prepared for immunoblot analysis using anti-cPLA(2) antibody. The results indicated that EC lysates contained cPLA(2) and that about 80% of the enzyme was unphosphorylated in unstimulated cells (Fig. 5, B and C). After 12.5 min of agonist stimulation more than 90% of the cPLA(2) was phosphorylated. The phosphorylation state was transient, and a return to near-base-line levels was apparent after 30 min. Densitometric analysis showed that the time course of phosphorylation of cPLA(2) was delayed slightly compared with that of p42 MAP kinase (Fig. 5C). The stimulation of cPLA(2) phosphorylation by basic FGF was concentration-dependent with a half-maximal phosphorylation at 0.5-1.0 ng/ml and near-maximal activity at 10 ng/ml (Fig. 6).


Figure 6: Concentration dependence of cPLA(2) phosphorylation in basic FGF-stimulated EC. Semi-confluent bovine aortic EC (25 times 10^6 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(2) antibody. The positions of unphosphorylated and phosphorylated cPLA(2) (cPLA-P) are indicated by large arrows.



Activation of cPLA(2) 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(2) activity occurred in response to basic FGF treatment of cell extracts or living cells. To specifically demonstrate cPLA(2) translocation, membrane and cytosolic fractions from basic FGF-treated EC were isolated and subjected to SDS-PAGE and immunoblotting with anti-cPLA(2) antibody (Fig. 7). These data clearly show that nearly all cPLA(2) is translocated to membranes within 12 min after stimulation of EC with basic FGF.


Figure 7: Basic FGF-stimulated translocation of cPLA(2) from cytosolic to membrane fractions. Semi-confluent EC cultures (25 times 10^6 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(2).



The Effect of Basic FGF on Cellular [^3H]Arachidonate Release and Comparison with cPLA(2) Phosphorylation and EC Motility

To determine whether activation of cPLA(2) by basic FGF was accompanied by release of arachidonate in intact cells, EC were prelabeled with [^3H]arachidonate, and agonist-induced release was measured after 30 min as accumulation of radiolabeled fatty acid in medium containing fatty acid-free albumin as a sink. In a representative experiment, 10 ng/ml basic FGF increased arachidonate release by more than 2-fold (143 cpm/well by control EC compared with 328 cpm/well by FGF-treated EC), whereas 1 µM PMA was much less effective (198 cpm/well) and 30 units/ml bovine thrombin was essentially inactive in these cells (162 cpm/well).

The in vitro PLA(2) assay results suggested that essentially all basic FGF-stimulated PLA(2) activity was due to activation of cPLA(2). In a dose-response experiment, maximal stimulation of basic FGF-induced [^3H]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(2) phosphorylation (Fig. 8). This result is consistent with the observation that complete phosphorylation of cPLA(2) 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(2) 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(2) activity is due to cPLA(2), suggest that cPLA(2) 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(2), [^3H]arachidonate release, and EC migration. Bovine aortic EC were prelabeled by incubation with [^3H]arachidonate (0.5 µCi/ml) for 24 h. The cells were then stimulated with basic FGF for 15 min and release of [^3H]arachidonate into the medium was measured (- - -- - -); unstimulated [^3H]arachidonate release was 32.3 fmol/well. Basic FGF-stimulated phosphorylation of cPLA(2) was quantitated by densitometry of the slow moving band in the Western blot shown in Fig. 6(-box-, relative densitometric units). Cell migration was measured as the number of cells crossing the wound line during a 22-h incubation (- - -bullet- - -); 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) .




DISCUSSION

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(2). The activated enzyme has biochemical and enzymatic characteristics consistent with cPLA(2), namely, a requirement for µM Ca and dithiothreitol insensitivity. Furthermore, basic FGF-stimulated PLA(2) activity in EC lysates is almost completely blocked by cPLA(2)-specific antiserum. The mechanism of activation is also consistent with cPLA(2), 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(2) in EC.

Basic FGF is far from unique in its activation of cPLA(2), 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(2) activity is due to activation of cPLA(2). This specificity is not universal, for example, anti-cPLA(2) antibody inhibits only half of the PLA(2) 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(2) as well as a distinct, unidentified PLA(2)(35) . We have also found that basic FGF by itself stimulates PLA(2) 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(2) 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(2)(38) . Since thrombin mobilizes EC calcium fluxes (39) , our finding that it is an ineffective agonist of PLA(2) 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(2) 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(2). 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(2), since granulocyte/macrophage colony-stimulating factor activates MAP kinase, but is a rather weak activator of cPLA(2)(31) . An additional activity required for full activation of cPLA(2) may be protein kinase C, which activates cPLA(2) 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(2) has been clearly shown. Diacylglycerol-mediated activation of protein kinase C and IP(3)-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(2) is not known. Finally, there is evidence for a role of guanine nucleotide-binding proteins (G-protein) in activation of PLA(2)(46) , and most likely, cPLA(2)(47, 48) (but G-protein-independent activation of cPLA(2) 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(2) activity. The identification of cPLA(2)-coupled G-proteins, the precise mechanisms by which these and other pathway components regulate cPLA(2) activity, and the role that cPLA(2) has in regulation of EC motility and other cellular functions are important questions that require further investigation.


FOOTNOTES

*
This work was supported in part by Grants HL40352, HL41178, and HL29582 (to P. L. F.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the American Heart Association, Northeast Ohio.

Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Cell Biology, Cleveland Clinic Research Institute, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-8053; Fax: 216-444-9404.

(^1)
The abbreviations used are: FGF, fibroblast growth factor; PLA(2), phospholipase A(2); cPLA(2), cytosolic phospholipase A(2); EC, endothelial cell(s); G-protein, guanine nucleotide-binding protein; MAP kinase, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.

(^2)
Y. Ivashchenko, unpublished data.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Michael Weber for providing anti-p42 MAP kinase antibody, Dr. Alan Wolfman for assistance with the methods to immunodeplete cell lysates of MAP kinase, and Dr. Bryan Williams for helpful discussions.


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