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J. Biol. Chem., Vol. 278, Issue 44, 43831-43837, October 31, 2003
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¶
From the
Department of Physiology and the Center for Vascular Biology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Received for publication, February 11, 2003 , and in revised form, August 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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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 (11–19). Phospholipase A2s (PLA2s) are the major rate-limiting enzymes in the release of arachidonic acid in many cell types (11, 20, 21). Among the growing number of PLA2s that have been isolated and characterized thus far, a calcium-dependent high molecular mass cytosolic PLA2 (cPLA2) and a calcium-independent PLA2 (iPLA2) 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 cPLA2 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 iPLA2, 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 iPLA2 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 iPLA2 in thrombin-induced VSMC proliferation. Here, we report for the first time that iPLA2 plays a predominant role in thrombin-induced arachidonic acid release and DNA synthesis and that these responses are mediated by p38 MAPK in VSMC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-32 P]ATP (3000 Ci/mmol), and [3H]thymidine (20 Ci/mmol) were obtained from PerkinElmer Life Sciences. A cell proliferation assay kit (MTT assay kit; 1465007) was from Roche Diagnostics. Phosphorothioate antisense (5'-GAGGCGTCCAAAGAACTGCAT-3') and sense (5'-ATGCAGTTCTTTGGACGCCTC-3') oligonucleotides of rat iPLA2 were made by IDT, Inc. (Coraville, IA). 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 µg/ml streptomycin. Cultures were maintained at 37 °C in a humidified 95% air and 5% CO2 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.
iPLA2 Assay—iPLA2 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 µlof50mM 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 µg of protein in a total volume of 45 µl from control and each treatment were added to microtiter plate wells consisting of 5 µl of assay buffer (80 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM CaCl2, 4 mM Triton X-100, 30% glycerol, and 1 mg/ml bovine serum albumin) with and without 10 µM BEL. The reaction was initiated by the addition of 200 µl of arachidonoyl thiophosphatidylcholine dissolved in 2x assay buffer and incubated at room temperature for 60 min. The reaction was then terminated by the addition of 10 µl of 25 mM 5,5'-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 iPLA2 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 5,5'-dithio-bis(2-nitrobenzoic acid) extinction coefficient value of 10.66 mM. The actual extinction coefficient for 5,5'-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).
[3H]Arachidonic or Oleic Acid Release—VSMC were labeled with [3H]arachidonic or oleic acid (0.3 µCi/ml) while growing exponentially, and at 90% confluence, cells were quiesced in DMEM containing 0.1% calf serum and 0.2 µCi/ml of the respective 3H-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 [3H]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 iPLA2 on thrombin-induced arachidonic acid release, cells were first labeled with [3H]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 µCi/ml [3H]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 µM leupeptin, 1 mM Na3VO4, and 400 µM 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 µl 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 MgCl2, 2 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM Na3VO4). The activity present in the immunoprecipitates was determined by resuspending the immunocomplex beads in 30 µl of kinase buffer containing 5 µg of myelin basic protein substrate, 20 µM ATP, and 1 µCi of [32P]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 x 103 cells/well in 100 µl of DMEM containing 10% fetal bovine serum and grown in a humidified incubator (95% air-5% CO2) 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 µl 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% CO2) 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 µl of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/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% polyacrylamide 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, iPLA2 activity, p38 MAPK activity and MTT assay are presented as mean ± S.D. The treatment effects were analyzed by Student's t test. p values < 0.05 were considered to be statistically significant. In the case of Western blot analysis, one representative set of data is shown.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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To test whether iPLA2 plays a role in thrombin-induced growth, we used both pharmacological and antisense oligonucleotide approaches. Inhibition of iPLA2 activity by BEL reduced thrombin-induced growth as measured by both [3H]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 iPLA2 levels by antisense oligonucleotides also blocked thrombin-induced DNA synthesis (Fig. 3D). Sense oligonucleotides of iPLA2 had no effect on thrombin-induced DNA synthesis. These results show that iPLA2 plays a role in thrombin-induced growth in VSMC.
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Earlier studies by other investigators have shown that p38 MAPK plays a role in thrombin-induced cPLA2 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 iPLA2 activity in VSMC, the role of p38 MAPK was 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 [-32P]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 iPLA2 activity, quiescent VSMC were treated with and without thrombin (0.5 unit/ml) in the presence and absence of 10 µM SB203580 or SB202190 for 30 min, and cell extracts were prepared. Equal amounts of protein from control and from each treatment were assayed for iPLA2 activity. Both SB203580 and SB202190 suppressed thrombin-stimulated iPLA2 activity by 70% (Fig. 5). Inhibition of p38 MAPK also blocked thrombin-induced growth in VSMC (Fig. 6, A and B).
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The important findings of the present study are as follows. 1) Thrombin stimulates iPLA2 activity in VSMC. 2) Thrombin-induced arachidonic acid release and growth are mediated by iPLA2. 3) Thrombin-induced iPLA2 activity, arachidonic acid release and DNA synthesis are dependent on p38 MAPK. A large body of data indicates that cPLA2 plays an important role in agonist-induced arachidonic acid release (11–13, 24). In addition, the agonist-induced acute activation of cPLA2 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 cPLA2 in serum and PDGF-BB-induced VSMC growth has been demonstrated (27, 28). Some studies have also shown that cPLA2-dependent arachidonic acid release is involved in apoptosis (19, 38). Besides cPLA2, the other PLA2 that is present in many cell types and involved in agonist-induced arachidonic acid release is the most recently characterized one, iPLA2 (21–23). One of the major functions attributed to iPLA2 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 iPLA2 (23). As in the case of cPLA2, the activity of iPLA2 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 iPLA2 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 iPLA2 activity by BEL, antisense oligonucleotides and p38 MAPK inhibitors suppresses the thrombin-induced growth in VSMC. A role for iPLA2 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 iPLA2 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 iPLA2, 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 adhesions (43–46). Because both cPLA2 and iPLA2 appear to be involved in agonist-induced arachidonic acid release (11, 20, 23–25, 29, 30) and a role for both PLA2s has been demonstrated in the regulation of cell growth (27, 28, 31), it would be interesting to learn which of these PLA2s 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 iPLA2 plays a predominant role in thrombin-induced arachidonic acid release and DNA synthesis, responses that are mediated by p38 MAPK in VSMC.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7321; Fax: 901-448-7126; E-mail: grao{at}physio1.utmem.edu.
1 The abbreviations used are: VSMC, vascular smooth muscle cells; PLA2, phospholipase A2; iPLA2, calcium-independent PLA2; cPLA2, cytosolic PLA2; PDGF, platelet-derived growth factor; BEL, bromoenol lactone; p38 MAPK, p38 mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Mops, 4-morpholinepropanesulfonic acid; ATF-1, activating transcription factor-1; CREB, cAMP-response element-binding protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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