Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells

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Biochi~ic~a et Biophysica A~ta Biochimica et Biophysiea Acta 1265 (1995) 67-72

Hydrogen peroxide activation of cytosolic phospholipase A 2 in vascular smooth muscle cells G.N. Rao *, M.S. Runge, R.W. Alexander Cardiology Division, Emory University School of Medicine, Atlanta, GA, USA Received 17 May 1994; revised 2 September 1994; accepted 3 October 1994

Abstract

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A 2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A 2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A 2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A 2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that eytosolic phospholipase A 2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase. Keywords: Arachidonic acid; cPLA2; Oxidant; Phosphorylation; Smooth muscle cell

1. Introduction

We have demonstrated in earlier studies that hydrogen peroxide, a reactive oxygen :species and a cellular oxidant, stimulates arachidonic acid release from prelabeled vascular smooth muscle cells (VSMC) and that the arachidonic acid mediates the hydrogen peroxide-induced c-los and c-jun mRNA expression [1,2]. We have also shown that the hydrogen peroxide-stimulated arachidonic acid release, and c-los and c-jun mRNA expression, can be blocked by mepacrine, a non-specific phospholipase A 2 (PLA 2) inhibitor, raising the possibility of PLA 2 action in these events [1,2]. PLA2s are a group of enzymes that hydrolyze the sn-2-acyl ester bond of phospholipids, generating free fatty acids and lysophopholipids [3-5]. Several low molecular mass (14-20 kDa), secretory type PLA2s (sPLA2s) have been purified and cloned [6,7]. Major features of sPLA2s

* Corresponding author. Present address: Cardiology Division, University of Texas Medical Branch, 9.142 Medical Research Building, 301 University Boulevard, Route 1064, Galveston, TX 77555-1064, USA. Fax: + 1 (409) 7470692. 0167-4889/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

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include their sensitivity to sulfhydryl reagents [6,8], their requirement for millimolar concentrations of calcium [6,7] and their lack of specificity for substrates with arachidonic acid in the sn-2 position [9]. Recently, however, several groups have identified, purified and cloned a high molecular mass cytosolic PLA 2 (cPLA 2) [10-15]. This cPLA 2 is expressed in a variety of cell types including fibroblasts, macrophages, and mesangial cells [10-15]. Furthermore, this cPLA 2 is mainly cytosolic [10-15], is active at physiological concentrations of calcium [16,17], is specific for substrates with arachidonic acid at the sn-2 position [16] and is regulated by phosphorylation and dephosphorylation [18-23]. It has been reported that arachidonic acid and its oxygenated metabolites play an important role in an array of biological processes such as signal transduction [24], chemotaxis [25], contraction [26,27] and cell growth and differentiation [28-34]. Given the substrate specificity of cPLA e and the pleiotrophic effects of arachidonic acid, we hypothesized that cPLA 2 may be important in cellular signaling and perhaps may transduce the hydrogen peroxide-elicited signals in VSMC. The purpose of the present study was, therefore, to examine whether hydrogen peroxide activates cPLA 2 in VSMC. In this communication we demonstrate for the first time that hydrogen peroxide

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stimulates phosphorylation of c P L A 2 in VSMC and protein kinase C, and mitogen activated protein kinase may mediate this effect.

2. Materials and methods 2.1. Materials

Aprotinin, arachidonic acid, benzamidine, bovine myelin basic protein, calmidazolium, EDTA, EGTA, fl-glycerophosphate, leupeptin, pepstatin A, phorbol 12,13-dibutyrate, phorbol 12-myristate 13-acetate, phosphoserine, phosphothreonine, phosphotyrosine, PMSF and sodium orthovanadate (Na3VO 4) were obtained from Sigma (St. Louis, MO). 5,6,8,9,11,12,14,15 [3H]arachidonic acid (221 Ci/mmol) and [32P]orthophosphoric acid (8500 Ci/mmol) were purchased from NEN (Boston, MA). [y-32p]ATP (5000 Ci/mmol) was from Amersham (Arlington Heights, IL). ERK-1 and ERK-2 anti-rabbit antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA). 2.2. Cell culture

VSMC were isolated from the thoracic aortae of 200250 g male Sprague-Dawley rats by enzymatic dissociation as described elsewhere [35]. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% ( v / v ) heat-inactivated calf serum, 100 U / m l penicillin and 100 /xg/ml streptomycin. Cultures were maintained in humidified 95% air/5% CO 2 at 37°C by passage of 1-3 • 105 cells/ml on reaching confluence. For all experiments, cells at 70-80% confluence were made quiescent by incubation for 48 h in fresh DMEM containing 0.1% calf serum. Cells were used at passage numbers 8-18. 2.3. [ 3H]Arachidonic acid release

VSMC were grown for 24 h in DMEM containing 10% serum and 0.5 /.LCi/ml [3H]arachidonic acid [36]. Cells were then growth-arrested by incubation for 48 h in fresh DMEM containing 0.1% serum and 0.5 /zCi/ml [3H]arachidonic acid. To initiate the experiment, cells were rinsed four times with, and incubated at 37°C in, 1 ml DMEM with or without agent. At the indicated times, the medium was removed and extracted with two volumes of chloroform/methanol (1:1). The organic phase was evaporated under nitrogen, resuspended in 200 /zl chloroform, spotted on Whatman silica gel 60A LK6D TLC plates and developed with the organic phase of the mixture ethyl acetate/hexane/acetic acid/water (85:35:15:90 v / v ) . 1 /zM unlabeled arachidonic acid standard was added as a carrier. The spot corresponding to arachidonic acid was visualized with iodine vapor, scraped, and its radioactivity measured by liquid scintillation spectrometry.

2.4. Phosphorylation of cytosolic

PLA 2

Growth-arrested VSMC were labeled with 3 0 0 / z C i / m l [32p]orthophosphate for 3 h at 37°C in Hepes-buffered salt solution (130 mM NaC1, 5 mM KCI, 1 mM MgC12, 1.5 mM CaC12, 20 mM Hepes (pH 7.4)) before exposure to 200 /xM hydrogen peroxide. At the end of the stimulation period, cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 15 min in 1.0 ml of 150 mM NaCI, 1% SDS, 50 mM Tris-HC1 (pH 7.5), 1 mM sodium orthovanadate, 0.5 mM PMSF, 50 mM NaF, 2 mM sodium pyrophosphate, 80 mM /3-glycerophosphate and 2 mM EGTA. The cell lysates were then clarified by centrifugation for 30 min at 14 000 rpm in a microfuge at 4°C. Cell lysates containing equal amounts of TCA-precipitable cpm were then incubated with 10 ~1 of polyclonal cPLA 2 antiserum overnight at 4°C. 50 /xl of protein A-Sepharose bead suspension in water (50% v / v ) were added to the samples and incubated for 90 min on ice with rocking. The beads were then collected by centrifugation at 6000 rpm for 1 min at 4°C in a microfuge, washed five times with ice-cold lysis buffer and once with ice-cold PBS. The immunoprecipitated proteins were dissolved by heating the beads at 100°C for 5 min in 40/zl Laemmli sample buffer and were separated by electrophoresis on 0.1% SDS, 10% polyacrylamide gels under reducing conditions [37]. The gels were dried and exposed to Kodak X-Omat AR X-ray film with an intensifying screen at - 7 0 ° C for 2 days. 2.5. Phosphoamino acid analysis

Cells were treated as described above for measurement of cPLA 2 phosphorylation. Following cell lysis, immunoprecipitation and SDS-PAGE, proteins were transferred to a PVDF membrane (Immobilon, Millipore, Bedford, MA) by electroblotting overnight at 100 mA [38]. After transfer, the membrane was autoradiographed. The cPLA 2 band was excised and exposed to vapors of 5.7 M HCI at ll0°C for 4 h in a reactivial (Pierce, Rockford, IL). After acid hydrolysis, one drop of methanol was placed on the membrane patches and amino acids and peptide fragments were extracted with 1 ml of water [39]. A mixture of unlabeled phosphoserine, phosphothreonine and phosphotyrosine (100 nmol each) was added to the aqueous extract before lyophilization over NaOH. Samples were dissolved in 20 /xl of water and spotted on a 20 × 20 cm cellulose thin layer chromatography plate (J.T. Baker, Phillipsburg, NJ). The phosphoamino acids were separated by one-dimensional electrophoresis in 7.8% acetic acid and 2.2% formic acid (pH 1.9) at 500 V for 4 h. Standards were visualized with ninhydrin before autoradiography. 2.6. Western blot analysis

Growth-arrested VSMC were incubated at 37°C for various times in the presence and absence of 200 /xM

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G.N. Rao et al. / Biochimica et Biophysica Acta 1265 (1995) 67-72

hydrogen peroxide. After incubation, medium was aspirated, cells rinsed with cold PBS and frozen immediately in liquid nitrogen. Cells were then thawed in 250 ~1 of lysis buffer (50 mM Hepes (pH 7.4), containing 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 2 mM sodium orthovanadate, 0.5 mM PMSF, 10 /zg/ml leupeptin and 0.01% Triton X-100) and sonicated. Cell lysates were cleared by centrifugation at 14000 rpm for 20 min in a microfuge. Cell lysates containing equal amounts of proteins from each condition were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. The membranes were probed with 1 /zg/ml of ERK-1 and ERK-2 anti-rabbit primary antibodies. After treating the membrane with peroxidase conjugated goat anti-rabbit secondary antibodies, peroxidase activity was detected using ECL reagents (Amersham, Arlington Heights, IL). 2.7. Kinase assay

Cytosolic extracts containing equal amounts of proteins from each condition were incubated in a final volume of 50/zl containing 50 mM fl-glycerophosphate (pH 7.3), 1.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol, 10/~M calmidazolium, 10 mM MgCI2, 10/~g/ml leupeptin, 10 /zg/ml aprotinin, 2 /.Lg/ml pepstatin A, 1 mM benzamidine, 0.3 mM [y-32p]ATP (sp. act. 2000 cpm/pmol) and 0.5 m g / m l myelin basic protein (MBP) for 20 rain at 37°C [40]. Reactions were terminated by the addition of 50/zl of cold 20% TCA. 50/~1 of the mix was then spotted on P-81 phosphocellulose filter. The filter was washed four times (5 min each) in 0.5% phosphoric acid and once with absolute ethanol. The filter was dried and the radioactivity was measured as described above. Densitometric analysis of the autoradiograms exposed in the linear range of film density was made on an LKB laser densitometer. All experiments were performed at least twice, with similar results unless otherwise indicated.

3. Results and discussion

We reported earlier that hydrogen peroxide induces arachidonic acid release from prelabeled VSMC and the arachidonic acid mediates hydrogen peroxide-stimulated protooncogene expression [1,2]. The present investigation sought to identify the enzyme responsible for hydrogen peroxide-induced release of arachidonic acid in VSMC. Several studies have demonstrated that cPLA 2 is specific for substrates of membrane phospholipids with arachidonic acid at the sn-2 position 1111-16]. In addition, it was demonstrated that growth factors and hormones activate c P L A 2 via its phosphorylafion [18-23]. To test whether hydrogen peroxide-stimulated release of arachidonic acid is mediated by cPLA2, the effect of hydrogen peroxide on

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Fig. 1. Time-course of hydrogen peroxide-induced phosphorylation of cPLA 2. Growth-arrested VSMC were labeled with [32P]orthophosphate and exposed to 200 /zM hydrogen peroxide for the indicated times. Aliquots of cell lysates containing equal amounts of TCA-precipitable cpm were incubated with polyclonal cPLA 2 antiserum. The immunoprecipitates were separated on a 0.1% SDS, 10% polyacrylamide gel. 32P-labeled cPLA 2 was identified by its apparent molecular mass ( = 97 kDa) on the autoradiogram and is similar to that reported in Rat-2 cells [18]. Results shown are representative of one experiment performed twice.

phosphorylation was determined using polyclonal antibodies. Hydrogen peroxide stimulated a rapid and time-dependent increase in the phosphorylation of c P L A 2 (Fig. 1). Increases (0.8-fold) in the phosphorylation of c P L A 2 by hydrogen peroxide were evident at 10 min and reached a maximum of 3-fold by 30 min. To find whether the time-course of hydrogen peroxide-induced phosphorylation of c P L A 2 correlates temporally with arachidonic acid release, [3H]arachidonie acid-prelabeled, growth-arrested VSMC were exposed to 200 /~M hydrogen peroxide for varying times and [3H]arachidonic acid release in the medium was measured. Hydrogen peroxide caused time-dependent increases in arachidonic acid release (Fig. 2). Together, these results suggest that c P L A 2 accounts, at least in part, for arachidonic acid release induced by hydrogen peroxide in VSMC. Previous studies from other laboratories as well as ours demonstrated that phosphorylation of c P L A 2 by growth factors and hormones occurs on serine residue(s) [18,2123]. To determine the amino acid residue of c P L A 2 phosphorylated by hydrogen peroxide, phosphoamino acid analysis of cPLA 2 was performed. Serine was found to be the only amino acid residue of cPLA 2 phosphorylated by hydrogen peroxide (Fig. 3). This result is consistent with earlier findings on serine phosphorylation of cPLA 2 in response to growth factors and hormones [18,21-23], and suggests that hydrogen peroxide may share the kinase cascade of activities with the above agonists to stimulate this enzyme. Numerous studies indicated that PKC plays an important role in agonist-induced release of arachidonic acid [12,19,41,42]. To gain insight into the kinase cascade cPLA 2 cPLA 2

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Fig. 2. Time-course of hydrogen peroxide-induced release of [3H]arachidonic acid. VSMC were labeled with [3H]arachidonic acid and growth-arrested before exposure to vehicle (©) or 200 /~M hydrogen peroxide ( 0 ) for the indicated times. [3H]arachidonic acid released in the medium was separated and identified by thin layer chromatography. Points represent the mean + S.D. of values from two independent experiments performed in duplicate.

leading to the activation of c P L A 2 by hydrogen peroxide, we examined the role of PKC, a major agonist-responsive serine-threonine protein kinase, in this event. PKC was down-regulated by exposure of VSMC to 1 /zM phorbol 12,13-dibutyrate (PDBu) for 24 h. We and others have previously shown that this regimen is sufficient to deplete cellular PKC levels significantly ( > 90%) in VSMC and other cell types [12,23,43]. Growth-arrested, PKC-depleted VSMC were labeled with [32 P]orthophosphate for 4 h and treated with and without 200 /xM hydrogen peroxide for 30 min and cell lysates prepared. Cell lysates containing equal amounts of counts/min from control and hydrogen peroxide-treated VSMC were immunoprecipitated with p o l y c l o n a l c P L A 2 antibodies and the immunoprecipitants were analyzed by SDS-PAGE. As shown in Fig. 4, PKC depletion significantly (50%) reduced the hydrogen peroxide-induced phosphorylation of cPLA 2. To determine whether PKC depletion also results in diminished release of arachidonic acid, [3H]arachidonic acid-prelabeled, growth-arrested, PKC-depleted VSMC were treated with

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Fig. 4. PKC dependence of hydrogen peroxide-induced phosphorylation of cPLA:. Growth-arrested, PKC-depleted VSMC were labeled with [32p]orthophosphate and exposed to 200 /xM hydrogen peroxide for 30 min. Cell lysates were treated as in Fig. 1 and cPLA 2 was separated by SDS-PAGE. Results shown are representative of one experiment performed twice.

and without 200 /xM hydrogen peroxide for 30 min and the [3H]arachidonic acid released in the medium was measured. PKC depletion completely blocked the hydrogen peroxide-induced release of arachidonic acid (Fig. 5). Similar results were obtained by use of the PKC inhibitor staurosporine (data not shown). These findings suggest that PKC plays an important role in hydrogen peroxide-induced phosphorylation of cPLA 2 and [3H]arachidonic acid release. Because hydrogen peroxide-stimulated phosphorylation of cPLA 2 temporally correlates with arachidonic acid release and PKC down-regulation reduces the responsiveness of these events to induction by hydrogen peroxide, it is likely that cPLA 2 plays a role in hydrogen peroxide-induced release of arachidonic acid. The present findings are consistent with those of others [18,19,22] in macrophages, CHO cells and HL60 cells where a correlation between

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Fig. 3. Phosphoamino acid analysis of cPLA 2. Phosphorylated cPLA 2 by hydrogen peroxide was immunoprecipitated, separated on SDS-PAGE, transferred to a PVDF membrane and acid hydrolyzed. Phosphoamino acids were separated by electrophoresis on a cellulose TLC plate. Standards were visualized with ninhydrin and 32P-labeled amino acids detected by autoradiography. Results shown are representative of one experiment performed twice.

Fig. 5. Role of PKC in hydrogen peroxide-induced release of [3H]arachidonic acid. [3H]arachidonic acid-prelabeled, growth-arrested, PKC-depleted VSMC were exposed to 200 /.tM hydrogen peroxide and [3H]arachidonic acid released in the medium was measured as described in Section 2. Results represent mean + S.D. obtained from three separate experiments.

G.N. Rao et al. / Biochimica et Biophysica Acta 1265

agonist-induced cPLA2 p h o s p h o r y l a t i o n and [3H]arachidonic acid release has been demonstrated. The present findings on the role of PKC in hydrogen peroxidestimulated c P L A 2 phosphorylation and [3H]arachidonic acid release are also in agreement with those of Lin et al. [18] and Xing and Mattera [19] in CHO cells and HL60 cells, respectively, where PI(C inhibition or down-regulation blocked the agonist-induced cPLA: phosphorylation and [3H]arachidonic acid release. In fact, it has also been demonstrated that PKC is able to phosphorylate and activate c P L A 2 in cell-free systems [20,21], although in one case PKC-mediated c P L A 2 phosphorylation did not result in a proportionate increase iin its activity [20]. It is of interest that PKC depletion attenuated hydrogen peroxidestimulated c P L A 2 phosphorylation by only 50% whereas it completely blocked the hydrogen peroxide-induced release of arachidonic acid. A likely explanation for this discrepancy is that both c P L A 2 and s P L A 2 contribute to hydrogen peroxide-induced release of arachidonic acid in a PKC-dependent manner. Indeed, Asaoka et al. [44] have demonstrated that PKC mediates sPLA: activation in lymphocytes in response to treatments with agonists. Mitogen-activated protein ( M A P ) kinase, a serine/threonine protein kinase, is reported to phosphorylate and activate cPLA 2 in cell-free systems [20,21] as well as in intact CHO cells [20]. Since hydrogen peroxide mimicked growth factors to phosphorylate c P L a 2 on serine residue(s), it was intriguing to examine whether MAP kinases play a role in hydrogen peroxide-stimulated phosphorylation of cPLA 2. To test this, we studied the effect of hydrogen peroxide on MAP kinase activation using two different approaches: (i) gel mobility shift assay, and (ii) [32p]orthophosphate incorporation into myelin basic protein (MBP). The first approach is based on the fact that the phosphorylated and activated form of MAP kinase protein migrates more slowly than the non-phosphorylated inactive form on SDS-PAGE [45]. The second approach is a conventional method of determining MAP kinase activity using a specific substrate such as MBP [40]. Growth-arrested VSMC were treated with and without 200 # M hydrogen peroxide for various times, cell extracts prepared and divided into two aliquots. One aliquot was used for Western blotting whereas the second one was used for MBP phosphorylation assay. As shown in Fig. 6, hydrogen peroxide caused a shift in the mobility of MAP kinase protein on SDS-PAGE and increased MBP phosphorylation as compared to controls. Angiotensin II, which was previously reported to activate MAP kinases in this cell system [46,47], also shifted the mobility of MAP kinase protein on SDS-PAGE and caused substantial increases in the phosphorylation of MBP (Fig. 6). Since cPLA 2 has been reported to be a substrate for MAP kinase, and hydrogen peroxide increased this kinase activity, it is likely that hydrogen peroxide-induced activation of c P L A 2 , at least in part, is mediated by MAP kinase. Phosphorylation and actiwttion of c P L A 2 by hydrogen

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Fig. 6. Effect of hydrogen peroxide or angiotensin II on MAP kinase activation. Growth-arrestedVSMC were exposed to vehicle, 200 /zM hydrogen peroxideor 100 nM angiotensinII for the indicatedtimes and cell lysates prepared. Cell lysates containing equal amounts of proteins from each condition were immunoblottedwith ERK-1 and ERK-2 antiserum (A), or assayedfor [32p]orthophosphateincorporationinto myelin basic protein (B). Results shown in panel A are representativeof one experiment (performedtwice) and in panel B are mean-I-S.D, of three separate experiments.

peroxide may have several implications in VSMC. It was reported that active oxygen (AO) species cause vasoconstriction [26]. Arachidonic acid and its metabolites were also reported to be potent vasoconstrictors [27]. Because cPLA 2 is specific for phospholipid substrates with arachidonic acid at the sn-2 position, and hydrogen peroxide, one of the AO species, stimulates c P L A 2 in VSMC, it is likely that cPLAE-dependent generation of arachidonic acid and its metabolites play a role in AO species-induced vasoconstriction. Future studies should determine whether hydrogen peroxide also increases production of arachidonic acid metabolites in VSMC. Several recent studies demonstrated that arachidonic acid and its metabolites play a role in growth factor-induced mitogenesis [28,29]. It was also reported that lipoxygenase-cytochrome P-450 monooxygenase-dependent metabolites of arachidonic acid stimulate growth in various cell types including VSMC [30-33]. We reported previously that hydrogen peroxide induces protooncogene expression and DNA synthesis in VSMC [48] and that arachidonic acid mediates these events [1,2]. As hydrogen peroxide-induced arachidonic acid release appears to be mediated by c P L A 2 , it is possible that this enzyme may mediate the hydrogen peroxide-stimulated growth events in VSMC.

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Acknowledgements We thank Dr. J.L. Knopf for providing us with cPLA 2 antiserum, and Jacqueline M. Brewer for preparing the manuscript. This work was supported in part by a grant-inaid from the American Heart Association-Georgia Affiliate to G.N.R.

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