A Requirement for Calcium-independent Phospholipase A2 in Thrombin-induced Arachidonic Acid Release and Growth in Vascular Smooth Muscle Cells*

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A2 (iPLA2). Without affecting its levels, thrombin increased iPLA2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A 2 (iPLA 2 ). Without affecting its levels, thrombin increased iPLA 2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA 2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA 2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA 2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA 2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.
Dedifferentiation and proliferation of vascular smooth muscle cells (VSMC) 1 is one of the contributing factors in vessel wall thickening that occurs during the pathogenesis of atherosclerosis and in restenosis following angioplasty (1). VSMC possesses the ability to undergo both hyperplastic and hypertrophic growth and many molecules that are produced at the site of vascular injury, including cytokines, eicosanoids, oxidants, and peptide growth factors, can influence these responses (1)(2)(3)(4)(5)(6)(7). Although earlier studies have demonstrated that functional interference of peptide growth factors such as platelet-derived growth factor-AA (PDGF-AA) and basic fibroblast growth factor-2 (bFGF-2) negated to some extent the growth of VSMC resulting in amelioration of restenosis in experimental animal models (3,8), therapeutic measures targeting inhibition of function of such single molecules are less appreciated as these lesions are multifactor-dependent (9,10). Because the increased growth of VSMC in atherosclerosis and restenosis is a result of the actions of many bioactive molecules that are produced at the site of vascular injury/inflammation, understanding the signaling events that are common, at least to major vascular mitogens, may lead to the development of more effective therapeutics against these vascular lesions.
Arachidonic acid, a polyunsaturated fatty acid, is released in response to a large number of bioactive molecules and is involved in the mediation of several important biological functions including vascular contraction/relaxation, cell proliferation/differentiation, and cell survival/apoptosis (11)(12)(13)(14)(15)(16)(17)(18)(19). Phospholipase A 2 s (PLA 2 s) are the major rate-limiting enzymes in the release of arachidonic acid in many cell types (11,20,21). Among the growing number of PLA 2 s that have been isolated and characterized thus far, a calcium-dependent high molecular mass cytosolic PLA 2 (cPLA 2 ) and a calcium-independent PLA 2 (iPLA 2 ) have been shown to play an important role in arachidonic acid release in response to a number of stimulants including receptor tyrosine kinase and G protein-coupled receptor agonists (20 -25). Earlier studies from several laboratories, including ours, have reported that cPLA 2 activity is regulated by phosphorylation in many cell types and is involved in the mediation of serum and PDGF-BB-induced proliferation in VSMC (12, 26 -30). In regard to iPLA 2 , although a recent study showed that it plays a role in the control of lymphocyte growth (31), the mechanism of regulation of its activity is unclear. As iPLA 2 also appears to be important in arachidonic acid release in response to various agonists, in our effort to understand the mechanism of regulation of its activity and to elucidate the common mitogenic signaling events of VSMC, we have studied the role of iPLA 2 in thrombin-induced VSMC proliferation. Here, we report for the first time that iPLA 2 plays a predominant role in thrombin-induced arachidonic acid release and DNA synthesis and that these responses are mediated by p38 MAPK in VSMC.

MATERIALS AND METHODS
Reagents-Aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, sodium orthovanadate, sodium deoxycholate, and leupeptin were purchased from Sigma. Bromoenol lactone (BEL) was obtained from Biomol (Plymouth Meeting, PA). Myelin basic protein substrate was from Calbiochem (San Diego, CA). Anti-iPLA 2 antibodies (160507) and an iPLA 2 assay kit were purchased from Cayman Chemicals (Ann Arbor, MI). Cell Culture-VSMC were isolated from the thoracic aortas of 200 -300-g male Sprague-Dawley rats by enzymatic dissociation as described earlier (6). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37°C in a humidified 95% air and 5% CO 2 atmosphere. Cells were quiesced by incubation in DMEM containing 0.1% calf serum for 72 h and used to perform the experiments unless otherwise stated.
iPLA 2 Assay-iPLA 2 activity was measured using a kit following the manufacturer's instructions (Cayman Chemicals, Ann Arbor, MI). After appropriate treatments, VSMC were scraped into 1.5-ml Eppendorf tubes and collected by centrifugation. The cell pellet was suspended in 500 l of 50 mM Hepes, pH 7.4, containing 1 mM EDTA and sonicated at duty cycle 30% speed for 30 s with 1-min interval for 10 times on ice (Branson Sonifier 450). Cell homogenates were cleared by centrifugation at 10,000 rpm for 15 min at 4°C. Supernatants containing 100 g of protein in a total volume of 45 l from control and each treatment were added to microtiter plate wells consisting of 5 l of assay buffer (80 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM CaCl 2 , 4 mM Triton X-100, 30% glycerol, and 1 mg/ml bovine serum albumin) with and without 10 M BEL. The reaction was initiated by the addition of 200 l of arachidonoyl thiophosphatidylcholine dissolved in 2ϫ assay buffer and incubated at room temperature for 60 min. The reaction was then terminated by the addition of 10 l of 25 mM 5,5Ј-dithio-bis(2-nitrobenzoic acid), 475 mM EGTA in 0.5 M Tris-HCl (pH 8.0), and the absorbance was measured at 414 nm in a SpectraMax190 microtiter plate reader (Molecular Devices, Sunnyvale, CA). To determine iPLA 2 activity, the optical density obtained in the presence of BEL was subtracted from the total optical density and the resulting optical density was converted into nanomoles of substrate hydrolyzed/min/ml using the 5,5Ј-dithiobis(2-nitrobenzoic acid) extinction coefficient value of 10.66 mM. The actual extinction coefficient for 5,5Ј-dithio-bis(2-nitrobenzoic acid) is 13.6 mM at 414 nm; this value has been adjusted for the path length of the solution in the well (manufacturer's instructions).
[ 3 H]Arachidonic or Oleic Acid Release-VSMC were labeled with [ 3 H]arachidonic or oleic acid (0.3 Ci/ml) while growing exponentially, and at 90% confluence, cells were quiesced in DMEM containing 0.1% calf serum and 0.2 Ci/ml of the respective 3 H-labeled fatty acid for 72 h at 37°C. Cells were then rinsed several times with DMEM. After rinsing, cells were added with 2 ml of DMEM containing 0.1% bovine serum albumin and treated with and without thrombin (0.5 unit/ml) in the presence and absence of the indicated pharmacological inhibitors for 30 min; then [ 3 H]arachidonic or oleic acid release into the medium was measured as described previously (28). In the case of testing the down-regulation of expression of iPLA 2 on thrombin-induced arachidonic acid release, cells were first labeled with [ 3 H]arachidonic acid and then transfected with antisense or sense oligonucleotides. After exposure to antisense or sense oligonucleotides for 72 h in serum-free DMEM, cell were treated with and without thrombin (0.5 unit/ml) for 30 min, and arachidonic acid release into the medium was measured as described above.
DNA Synthesis-VSMC with and without appropriate treatments were pulse-labeled with 1 Ci/ml [ 3 H]thymidine for the last 12 h of the 24-h incubation period. After labeling, cells were washed with cold phosphate-buffered saline (PBS), trypsinized, and collected by centrifugation. The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysate mixture was passed through a glass fiber filter (GF/C, Whatman). The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The filter was dried and placed in a liquid scintillation vial containing the scintillation fluid, and the radioactivity was measured in a Beckman liquid scintillation counter (model LS 5000TA).
p38 MAPK Activity-After appropriate treatments, cells were washed with cold PBS and lysed on ice for 15 min in lysis buffer containing 20 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 50 mM ␤-glycerophosphate, 1% Triton X-100, 10 units/ml aprotinin, 2 M leupeptin, 1 mM Na 3 VO 4 , and 400 M phenylmethylsulfonyl fluoride. The cell lysates were cleared by centrifugation at 12,000 rpm for 10 min at 4°C. Cell lysates normalized for protein were immunoprecipitated by incubation with anti-p38 MAPK rabbit IgG for 2 h followed by the addition of 40 l of 50% (w/v) protein A-Sepharose beads for an additional hour. The beads were washed three times with lysis buffer, three times with wash buffer (100 mM Tris-HCl, pH 7.6, 500 mM lithium chloride, 0.1% Triton X-100, and 1 mM dithiothreitol), and three times with kinase buffer (12.5 mM Mops, pH 7.5, 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 2 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM Na 3 VO 4 ). The activity present in the immunoprecipitates was determined by resuspending the immunocomplex beads in 30 l of kinase buffer containing 5 g of myelin basic protein substrate, 20 M ATP, and 1 Ci of [␥ 32 P]ATP per reaction and incubating at 30°C for 20 min. After incubation, the reaction mixture was spotted on P81 phosphocellulose paper. The filter paper was then washed three times with 0.75% phosphoric acid and one time with acetone. The filters were placed in scintillation vials containing the scintillation fluid, and the radioactivity was counted in a Beckman liquid scintillation counter (model LS 5000TA).
MTT Assay-VSMC growth was measured using a kit from Roche Molecular Biochemicals. VSMC were plated onto a 96-well tissue culture plate at a density of 2 ϫ 10 3 cells/well in 100 l of DMEM containing 10% fetal bovine serum and grown in a humidified incubator (95% air-5% CO 2 ) at 37°C. At about 80% confluence, cells were quiesced. Quiescent cells were then treated with and without thrombin (0.5 unit/ml) in the presence and absence of indicated inhibitors for 24 h. After treatments, 10 l of 0.5 mg/ml MTT labeling reagent in PBS was added to each well, and cells were incubated in a humidified incubator (95% air-5% CO 2 ) at 37°C for 4 h. One hundred microliters of solubilization solution (10% SDS in 0.01 M HCl) was then added, and incubation was continued overnight. Formazan, a dark blue product formed by the cleavage of MTT by living cells, was measured at 570 nm in a SpectraMax 190 microtiter plate reader (Molecular Devices).
Western Blot Analysis-After appropriate treatments, VSMC were rinsed with cold PBS and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 l of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 100 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM sodium orthovanadate) and scraped into 1.5-ml Eppendorf tubes. After standing on ice for 20 min, the cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell lysates containing equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% poly-FIG. 1. Thrombin stimulates iPLA 2 activity in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 unit/ml) for the indicated times, and cell extracts were prepared. Equal amounts of protein (30 g) from control and each treatment were either assayed for iPLA 2 activity using a commercially available kit (A) or analyzed by Western blotting using anti-iPLA 2 antibodies (B). *, p Ͻ 0.01 versus control.
acrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Biosciences). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).
Statistics-All experiments were repeated three times with similar results. Data on arachidonic and oleic acid release, DNA synthesis, iPLA 2 activity, p38 MAPK activity and MTT assay are presented as mean Ϯ S.D. The treatment effects were analyzed by Student's t test. p values Ͻ 0.05 were considered to be statistically significant. In the case of Western blot analysis, one representative set of data is shown.

RESULTS AND DISCUSSION
To understand the mitogenic signaling events of thrombin in VSMC, we have studied the role of iPLA 2 . Quiescent VSMC were treated with and without thrombin (0.5 unit/ml) for various times, and cell extracts were prepared. Equal amounts of protein from control and thrombin-treated VSMC were assayed for iPLA 2 activity using arachidonoyl thiophosphatidylcholine as a substrate. H]oleic acid released into the medium was measured. D, equal amounts of protein (30 g) from VSMC that were exposed to antisense or sense oligonucleotides of iPLA 2 or LipofectAMINE alone were analyzed by Western blotting for iPLA 2 levels using its specific antibodies. E, VSMC were transfected with antisense or sense oligonucleotides of iPLA 2 , prelabeled with [ 3 H]arachidonic acid, and subjected to treatment with and without thrombin (0.5 unit/ml) for 30 min followed by measurement of the [ 3 H]arachidonic acid released into the medium. *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus thrombin treatment alone. AS, antisense oligonucleotides; S, sense oligonucleotides. activity was observed in thrombin-treated VSMC compared with control (Fig. 1A). To find whether the observed increase in iPLA 2 activity in VSMC by thrombin was due to its increased production, we measured its steady-state levels. Western blot analysis of an equal amount of protein from control and thrombin-treated VSMC showed no apparent change in iPLA 2 levels over a 16-h treatment period (Fig. 1B). These results indicate that increases in iPLA 2 activity by thrombin were not due to its increased production. To learn the functional significance of activation of iPLA 2 by G protein-coupled receptor agonist, we next studied the effect of thrombin on arachidonic acid release. VSMC that were prelabeled with [ 3 H]arachidonic acid and quiesced were treated with and without thrombin (0.5 unit/ml) in the presence and absence of 10 M BEL, a selective inhibitor of iPLA 2 (32), for 30 min, and [ 3 H]arachidonic acid released into the medium was measured. Thrombin stimulated [ 3 H]arachidonic acid release by 2-3-fold, and 70% of this effect was suppressed by BEL ( Fig. 2A). A dose-response curve for BEL also showed that the optimum concentration of the drug at which it inhibits thrombin-induced [ 3 H]arachidonic acid release to a maximum level is 10 M (Fig. 2B). Earlier studies from other laboratories have reported that in addition to its mechanismbased inhibition of iPLA 2 activity, BEL suppresses phosphatidic acid phosphohydrolase activity (33) resulting decreased diacylglycerol production. Decreased production of diacylglycerol, in turn, by affecting protein kinase C (PKC) could lead to inhibition of agonist-induced arachidonic acid release. Therefore, to find whether BEL blocks thrombin-induced arachidonic acid release by inhibiting phosphohydrolase activity, we tested the effect of propranolol, a potent inhibitor of phosphohydrolase (34), on thrombin-stimulated arachidonic acid release in VSMC. As shown in Fig. 2A, propranolol (10 M) had no significant effect on thrombin-induced arachidonic acid release. This result indicates that BEL inhibits thrombin-induced arachidonic acid release independently of its affect on phosphohydrolase. Unlike cPLA 2 , iPLA 2 does not exhibit substrate specificity for arachidonic acid at the sn-2 position of phospholipids. Therefore, to obtain an additional line of evidence for the role of iPLA 2 in thrombin-induced arachidonic acid release, VSMC were prelabeled with [ 3 H]oleic acid, quiesced, and treated with and without thrombin (0.5 unit/ml) in the presence and absence of 10 M BEL for 30 min, and [ 3 H]oleic acid released into the medium was measured. If thrombin stimulates arachidonic acid release via activation of iPLA 2 , then one would expect that thrombin also induces oleic acid release in a manner that is sensitive to inhibition by BEL. Consistent with this expectation, thrombin induced [ 3 H]oleic acid release by about 3-fold, and this response was significantly blocked by BEL (Fig. 2C). To confirm the role of iPLA 2 in thrombin-induced arachidonic acid release, we next used an antisense oligonucleotide approach. VSMC were transfected with 5 g/ml antisense or sense oligonucleotides of iPLA 2 using LipofectAMINE reagent. Sev- enty-two hours after transfection, cell lysates were prepared and analyzed by Western blotting for iPLA 2 levels. Antisense oligonucleotides of iPLA 2 suppressed its levels by almost 80% as compared with levels in the LipofectAMINE-treated control (Fig. 2D). Sense oligonucleotides of iPLA 2 , on the other hand, did not affect its levels. VSMC that were transfected with antisense or sense oligonucleotides of iPLA 2 and prelabeled with [ 3 H]arachidonic acid were treated with and without thrombin (0.5 unit/ml) for 30 min, and the [ 3 H]arachidonic acid released into the medium was measured. Antisense but not sense oligonucleotides of iPLA 2 blocked thrombin-induced [ 3 H]arachidonic acid release (Fig. 2E). Together, these results clearly indicate a role for iPLA 2 in thrombin-induced arachidonic acid release in VSMC.
To test whether iPLA 2 plays a role in thrombin-induced growth, we used both pharmacological and antisense oligonucleotide approaches. Inhibition of iPLA 2 activity by BEL reduced thrombin-induced growth as measured by both [ 3 H]thymidine incorporation and MTT assay (Fig. 3, A and C). Consistent with its lack of effect on thrombin-induced arachidonic acid release, propranolol did not affect thrombin-induced DNA synthesis (Fig. 3B). Down-regulation of iPLA 2 levels by antisense oligonucleotides also blocked thrombin-induced DNA synthesis (Fig. 3D). Sense oligonucleotides of iPLA 2 had no effect on thrombin-induced DNA synthesis. These results show that iPLA 2 plays a role in thrombin-induced growth in VSMC.
Earlier studies by other investigators have shown that p38 MAPK plays a role in thrombin-induced cPLA 2 phosphorylation and activation in platelets (35). Recently, we have reported that p38 MAPK via phosphorylation and activation of ATF-1 is involved in thrombin-induced growth in VSMC (36). Therefore, to understand the possible mechanism(s) by which thrombin stimulates iPLA 2 activity in VSMC, the role of p38 MAPK was FIG. 4. Thrombin stimulates p38 MAPK activity in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 unit/ml) for the indicated times or for 30 min in the presence and absence of the indicated p38 MAPK inhibitors (10 M), and cell extracts were prepared. Equal amounts of protein from control and each treatment were either analyzed by Western blotting for p38 MAPK levels using its phosphospecific or normal antibodies (A) or subjected to immunoprecipitation with normal anti-p38 MAPK antibodies followed by determination of the kinase activity in the immunocomplexes using myelin basic protein and [␥-32 P]ATP as substrates (B). *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus thrombin treatment alone. tested. An equal amount of protein from control and various times of thrombin-treated VSMC was analyzed by Western blotting for phosphorylated levels of p38 MAPK using its phosphospecific antibodies. As shown in Fig. 4A, thrombin activated p38 MAPK in a time-dependent manner in VSMC as determined by an increase in its phosphorylation level. Thrombin also increased p38 MAPK activity as determined by immunocomplex kinase assay using myelin basic protein and [␥-32 P]ATP as substrates (Fig. 4B). In addition, SB203580 and SB202190, two structurally different and potent inhibitors of p38 MAPK (37), completely blocked thrombin-induced p38 MAPK activity (Fig. 4B). To find whether p38 MAPK plays a role in thrombin-stimulated iPLA 2 activity, quiescent VSMC were treated with and without thrombin (0.5 unit/ml) in the presence and absence of 10 M SB203580 or SB202190 for 30 min, and cell extracts were prepared. Equal amounts of protein from control and from each treatment were assayed for iPLA 2 activity. Both SB203580 and SB202190 suppressed thrombinstimulated iPLA 2 activity by 70% (Fig. 5). Inhibition of p38 MAPK also blocked thrombin-induced growth in VSMC (Fig. 6,  A and B).
The important findings of the present study are as follows. 1) Thrombin stimulates iPLA 2 activity in VSMC. 2) Thrombin-induced arachidonic acid release and growth are mediated by iPLA 2 . 3) Thrombin-induced iPLA 2 activity, arachidonic acid release and DNA synthesis are dependent on p38 MAPK. A large body of data indicates that cPLA 2 plays an important role in agonist-induced arachidonic acid release (11)(12)(13)24). In addition, the agonist-induced acute activation of cPLA 2 requires its phosphorylation, and it is mediated by serine/threonine kinases such as PKC, MAPKs, and calcium-/ calmodulin-dependent kinase II (11-13, 24, 26, 29, 30). Furthermore, the involvement of cPLA 2 in serum and PDGF-BBinduced VSMC growth has been demonstrated (27,28). Some studies have also shown that cPLA 2 -dependent arachidonic acid release is involved in apoptosis (19,38). Besides cPLA 2 , the other PLA 2 that is present in many cell types and involved in agonist-induced arachidonic acid release is the most recently characterized one, iPLA 2 (21-23). One of the major functions attributed to iPLA 2 is its role in lipid remodeling (39,40). In fact, using its selective inhibitors such as BEL, it was shown that arachidonic acid release in response to some agonists is mediated primarily by iPLA 2 (23). As in the case of cPLA 2 , the activity of iPLA 2 in response to some agents such as phorbol 12-myristate 13-acetate has been reported to be regulated by a novel PKC isoform, PKC-⑀ (41). In this aspect, the present study provides additional evidence that iPLA 2 activity is regulated by serine/threonine kinases, particularly p38 MAPK, in VSMC in response to thrombin. In addition, the present study shows that blockage of iPLA 2 activity by BEL, antisense oligonucleotides and p38 MAPK inhibitors suppresses the thrombin-induced growth in VSMC. A role for iPLA 2 in lymphocyte growth has also been reported (31). Previously, we showed that p38 MAPK plays a role in thrombin-induced VSMC growth via activation of ATF-1 (36). Recently it was demonstrated that iPLA 2 plays a role in double stranded-RNA-induced nitric oxide synthase gene expression via activation of CREB (42). Because CREB/ ATF-1 mediate cAMP-response element-dependent gene expression, one possible mechanism by which p38 MAPK is involved in thrombin-induced growth in VSMC is via activation of iPLA 2 , thereby releasing arachidonic acid, which in turn stimulates ATF-1-dependent gene expression. Previous work from other laboratories showed that arachidonic acid and its eicosanoid metabolites are involved in the mediation of mitogenic signaling events such as formation of focal ad-hesions (43)(44)(45)(46). Because both cPLA 2 and iPLA 2 appear to be involved in agonist-induced arachidonic acid release (11, 20, 23-25, 29, 30) and a role for both PLA 2 s has been demonstrated in the regulation of cell growth (27,28,31), it would be interesting to learn which of these PLA 2 s is involved in receptor tyrosine kinase and G protein-coupled receptor agonist-induced focal adhesion formations.
In summary, the present study demonstrates for the first time that iPLA 2 plays a predominant role in thrombin-induced arachidonic acid release and DNA synthesis, responses that are mediated by p38 MAPK in VSMC.