Involvement of Phosphatidylcholine-Specific Phospholipase C in Thromboxane A2-Induced Activation of Mitogen-Activated Protein Kinase in Astrocytoma Cells

Involvement of Phosphatidylcholine-Specific Phospholipase C inThromboxane A2-Induced Activation of Mitogen-Activated

Protein Kinase in Astrocytoma Cells

*Hiroshi Kobayashi, *Shigeyoshi Honma, †Norimichi Nakahata, and *Yasushi Ohizumi

Departments of*Pharmaceutical Molecular Biology and†Cellular Signaling, Graduate School of Pharmaceutical Sciences,Tohoku University, Sendai, Japan

Abstract: Thromboxane A2 (TXA2) receptor-mediatedsignal transduction was investigated in 1321N1 humanastrocytoma cells. 9,11-Epithio-11,12-methano-TXA2(STA2), a TXA2 receptor agonist, induced Ca21 mobiliza-tion and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited bytreatment with U73122, an inhibitor of phosphatidylino-sitol-specific phospholipase C, or by culturing in 0.5%fetal calf serum containing 0.5 mM dibutyryladenosine39,59-cyclic monophosphate (dbcAMP) for 2 days. How-ever, the dbcAMP treatment augmented the TXA2 recep-tor-mediated phosphorylation of mitogen-activated pro-tein kinase (MAPK). These results were confirmed by afunctional MAPK assay measuring the incorporation of32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, aTXA2 receptor antagonist, and GF109203X, an inhibitor ofprotein kinase C. Although U73122 did not inhibit or onlyslightly inhibited the activation of MAPK, D-609, an inhib-itor of phosphatidylcholine-specific phospholipase C, po-tently attenuated the activation in a concentration-depen-dent manner. Furthermore, STA2 accelerated the releaseof [3H]choline metabolites from the cells prelabeled with[3H]choline chloride. This release was inhibited by treat-ment with D-609. These results suggest that phosphati-dylcholine-specific phospholipase C and protein kinaseC, but not phosphatidylinositol-specific phospholipaseC, are involved in TXA2 receptor-mediated MAPK activa-tion in 1321N1 human astrocytoma cells. Key Words:Phosphatidylcholine-specific phospholipase C—Proteinkinase C—Phosphoinositide hydrolysis—Astrocytomacells—Thromboxane receptor—Mitogen-activated pro-tein kinase.J. Neurochem. 74, 2167–2173 (2000).

Thromboxane A2 (TXA2), a metabolite of arachidonicacid, is known to induce many cellular responses, includ-ing platelet aggregation, vasoconstriction, and broncho-constriction, causing thrombosis, hypertension, andbronchial asthma. We previously reported that 1321N1human astrocytoma cells (Nakahata et al., 1989) andrabbit astrocytes (Nakahata et al., 1992) express TXA2

receptors. Astrocytes are known to be one of the targetcells of neurons, having the function of maintainingneurons by releasing several neurotrophic factors (Levi-Montalcini, 1987), by uptake of excess transmitters andions (Somjen, 1988), and by gliosis when the CNS isinjured. It has been reported that a dramatic increase ingeneration of arachidonic acid metabolites occurs inbrain following posthypoxic or postanoxic injury (Bazanand Rodriguez de Turco, 1980). In addition, the elevationof immunoreactivity to cytosolic phospholipase A2

(cPLA2) that initiates the arachidonic acid cascade isobserved in astrocytes in Alzheimer’s disease brain (Ste-phenson et al., 1996). Thus, arachidonic acid metabo-lites, including TXA2, might play important roles inbrain, particularly under pathological conditions.

The TXA2 receptor is coupled to a guanine nucleotidebinding protein (G protein) of the Gq/11 class and phospha-tidylinositol 4,5-bisphosphate-specific phospholipase C (PI-PLC) in 1321N1 human astrocytoma cells (Nakahata et al.,1995), activation of which results in inositol 1,4,5-trisphos-

Received September 7, 1999; revised manuscript received December13, 1999; accepted December 20, 1999.

Address correspondence and reprint requests to Dr. N. Nakahata atDepartment of Cellular Signaling, Graduate School of PharmaceuticalSciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: [email protected]

Abbreviations used:[Ca21]i, intracellular free Ca21 concentration;cPLA2, cytosolic phospholipase A2; D-609, carbonodithioic acid,o-(oc-tahydro-4,7-methano-1H-inden-5-yl) ester; dbcAMP, dibutyryladen-osine 39,59-cyclic monophosphate; DMEM, Dulbecco’s modifiedEagle’s medium; EMEM, Eagle’s minimum essential medium;FCS, fetal calf serum; G protein, guanine nucleotide binding pro-tein; GF109203X, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; MAPK, mitogen-activated pro-tein kinase; MEK, mitogen-activated protein kinase kinase; PC, phos-phatidylcholine; PC-PLC, phosphatidylcholine-specific phospholipaseC; PD98059, 29-amino-39-methoxyflavone; PI-PLC, phosphatidylino-sitol 4,5-bisphosphate-specific phospholipase C; PKA, protein kinaseA; PKC, protein kinase C; SQ29548, [1S-[1a,2b(5Z),3b,4a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenic acid; STA2, 9,11-epithio-11,12-methanothromboxaneA2; TXA2, thromboxane A2; U73122, 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

phate accumulation (Nakahata et al., 1989) and an increasein intracellular free Ca21 concentration ([Ca21]i) (Sakaiet al., 1996). The TXA2 receptor-mediated signaling forCa21 mobilization in 1321N1 human astrocytoma cells issimilar to that in platelets (Shenker et al., 1991; Baldassareet al., 1993; Knezevic et al., 1993; Ohkubo et al., 1996b).Recently, it has been shown that the TXA2 receptor alsocouples with G12 in human platelets (Offermanns et al.,1994) and Gh in human erythroleukemia and human aorticsmooth muscle cells (Vezza et al., 1999). Although thefunctions of these G proteins have not been elucidated, theTXA2 receptor may have multiple signal transduction path-ways.

Phospholipid degradation is induced by various stim-uli of cells and is involved in many cellular functions.Most of the work has focused on phosphatidylinositolturnover, but recent studies have shown the importanceof phosphatidylcholine (PC) hydrolysis (Exton, 1997). Ithas been reported that PC-specific phospholipase C (PC-PLC) is involved in the apoptotic signaling of tumornecrosis factor (Cifone et al., 1995) and in the mitogenicsignaling of platelet-derived growth factor (van Dijket al., 1997). D-609, a specific inhibitor of PC-PLC, hasbeen used as a tool for analyzing the activity of PC-PLC.

Mitogen-activated protein kinase (MAPK) is a serine/threonine kinase that exists ubiquitously in eukaryoticorganisms and regulates cell proliferation (Derijard et al.,1995), differentiation (Marshall, 1995), and many othercellular functions (Gotoh and Nishida, 1996). MAPK isa component of the serine/threonine kinase cascade,composed of ras, raf, and MAPK kinase (MEK). Thiscascade can be activated by various receptors, includingG protein-coupled receptors (Winitz et al., 1993; Kasuyaet al., 1994). It has been shown that TXA2 stimulates anMAPK cascade in vascular smooth muscles resulting incell growth (Morinelli et al., 1994). In platelets that arenot proliferative, TXA2 also stimulates the MAPK cas-cade resulting in cPLA2 activation and arachidonic acidrelease (Ohkubo et al., 1996a). Although both ras andprotein kinase C (PKC) have been shown to be involvedin activation of the MAPK pathway in various tissues orcells (Thomas et al., 1992; van Biesen et al., 1996), thedetailed mechanism of MAPK activation mediated viathe TXA2 receptor remains unknown.

It has been shown that dibutyryladenosine 39,59-cyclicmonophosphate (dbcAMP) induces morphological changesof astrocytes reflecting from cell differentiation (Lim et al.,1976; Honma et al., 1999) and augmentation of glutaminesynthetase activity (Nelson and Siman, 1990), a biochemi-cal marker for astroglial differentiation. Thus, cells treatedwith dbcAMP have been used as a good model of differ-entiated astrocytes.

The present study was undertaken to investigate themechanism of TXA2 receptor-mediated MAPK acti-vation in 1321N1 human astrocytoma cells differenti-ated with dbcAMP. We obtained the results that theTXA 2 receptor-mediated MAPK activation is medi-ated via the PC-PLC/PKC pathway but not the PI-PLC/Ca21 pathway.

MATERIALS AND METHODS

MaterialsDulbecco’s modified Eagle’s medium (DMEM) and Eagle’s

minimum essential medium (EMEM) were purchased fromNissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Fetal calfserum (FCS) was purchased from General Scientific Labora-tory (Los Angeles, CA, U.S.A). 9,11-Epithio-11,12-methano-TXA2 (STA2) was kindly given by Ono Pharmaceutical Co.,Ltd. (Tokyo). [1S-[1a,2b(5Z),3b,4a]]-7-[3-[[2-[(Phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenic acid (SQ29548) was given by Squibb Japan (Tokyo).Bovine serum albumin andN6,2-O-dibutyryladenosine 39,59-cyclic monophosphate (dbcAMP) were purchased from SigmaChemical Co. (St. Louis, MO, U.S.A.). Phospho-MAPK anti-body and 29-amino-39-methoxyflavone (PD98059) were pur-chased from New England Biolabs (Beverly, MA, U.S.A.).1-[6-[[(17b)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione (U73122), 3-[1-[3-(dimethyl-amino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (GF109203X), and carbonodithioic acid,o-(octahy-dro-4,7-methano-1H-inden-5-yl) ester (D-609) was fromFunakoshi Co. Ltd. (Tokyo). The Immun-Lite II chemilumi-nescence assay kit was from Bio-Rad (Hercules, CA, U.S.A.).The p42/p44 MAPK enzyme assay system, [g-32P]ATP, and[3H]choline chloride was from Amersham International (Buck-inghamshire, U.K.). Fura 2 acetoxymethyl ester was fromDojindo (Kumamoto, Japan). Other chemicals or drugs were ofreagent grade or the highest quality available.

Cell culture and differentiation1321N1 human astrocytoma cells were grown in DMEM

supplemented with 5% FCS, 50 units/ml penicillin, and 50mg/ml streptomycin (Sakai et al., 1996). Cells were maintainedin a humidified incubator in an atmosphere of 95% air and 5%CO2. For differentiation, the cells were cultivated for 2–3 daysin medium containing 0.5% FCS and 0.5 mM dbcAMP (dif-ferentiation medium).

Assay of inositol phosphatesPhosphoinositide breakdown was monitored by quantifying

3H-inositol phosphates as previously described (Nakahataet al., 1989). Cells (105/ml) in a six-well plate were cultured inDMEM supplemented with 5% FCS for 3 days and then ex-posed to differentiation medium for 3 days. Phosphoinositideswere labeled with DMEM containing [3H]inositol (2 mCi/ml)for 18–24 h. The cells were preincubated in EMEM bufferedwith 20 mM HEPES (EMEM/HEPES; pH 7.4) containing 10mM LiCl at 37°C for 10 min. The reaction was initiated byaddition of STA2 and was terminated by addition of 1 ml of 5%trichloroacetic acid after aspiration of the medium. For theexperiments using various inhibitors, they were preincubatedfor 10 min. The trichloroacetic acid extract was washed threetimes with diethyl ether to remove trichloroacetic acid, anddiethyl ether was removed by maintaining the sample at 47°Cfor 20 min. Total3H-inositol phosphates were separated on ananion exchange column (AG 1X-8, formate form). The trichlo-roacetic acid precipitate was dissolved in 0.5 ml of 1M NaOH,and the3H radioactivity was measured after neutralization forquantifying3H-labeled phosphoinositides.

Measurement of [Ca21]iThe change in [Ca21]i was measured by monitoring the

intensity of fura 2 fluorescence as described previously (Naka-hata et al., 1994).

J. Neurochem., Vol. 74, No. 5, 2000

2168 H. KOBAYASHI ET AL.

Western blottingCells (105/ml) in a six-well plate were cultured in DMEM

supplemented with 5% FCS for 3 days and then exposed todifferentiation medium for an additional 2 days. The cellswere preincubated in EMEM/HEPES (pH 7.4) for 10 min.The reaction was initiated by addition of STA2 and wasterminated by addition of 0.25 ml of Laemmli’s samplebuffer after aspiration of the medium. Western blotting wasperformed as described previously (Rho et al., 1997). Pro-tein was transferred to a polyvinylidene difluoride mem-brane and then incubated with anti-phospho-MAPK anti-body (2,500-fold dilution). Then the membranes were incu-bated with alkaline phosphatase-conjugated goat anti-rabbitIgG antibody (3,000-fold dilution) at 25°C for 2 h. Immu-noreactivity was determined by a chemiluminescence assaykit (Immun-Lite II; Bio-Rad) and visualized by exposing themembrane to ECL Hyper-film (Amersham).

MAPK assayCells (105/ml) in a 35-mm-diameter dish were cultured in

DMEM supplemented with 5% FCS for 2 days and then ex-posed to differentiation medium for an additional 2 days. Thecells were preincubated in EMEM/HEPES (pH 7.4) for 10 min.The reaction was initiated by addition of STA2 and was termi-nated by addition of 0.25 ml of ice-cold lysis buffer composedof 10 mM Tris, 2 mM EGTA, 2 mM dithiothreitol, 1 mMsodium orthovanadate, 1 mM ( p-amidinophenyl)methanesulfo-nyl fluoride, 10mg/ml leupeptin, 10mg/ml aprotinin, and 150mM NaCl (pH 7.4) after aspiration of the medium. For theexperiments using various inhibitors, they were preincubatedfor 10–20 min. After the reaction was terminated, cells werescraped and sonicated, and then the samples were centrifuged at15,000g for 10 min and incubated at 30°C for 30 min in a finalvolume of 30ml with buffer containing 2 mM substrate peptide,25 mM HEPES, 100 mM sodium orthovanadate, 0.017% so-dium azide, 0.1 mM [g-32P]ATP (200 mCi/ml), and 30 mMMgCl2, pH 7.4. After the incubation, the reaction was termi-nated by addition of 10ml of 300 mM orthophosphoric acid.The sample was spotted onto a phosphocellulose filter. Thefilter was washed twice with 1% acetic acid for 2 min and threetimes with distilled water for 2 min. The radioactivity remain-ing in the filter was counted with a liquid scintillation counter.Specific phosphorylation was calculated as the difference be-tween phosphorylation measured in the presence and absenceof substrate peptide.

Assay of PC hydrolysisCells (1.53 105 per well) in a six-well plate were cultured

in DMEM supplemented with 5% FCS for 3 days and thenexposed to differentiation medium for 2 days. The cells werelabeled with DMEM containing [3H]choline chloride (2mCi/ml) for 18–24 h. The cells were then washed twice and prein-cubated in incubating medium containing 118 mM NaCl, 4.7mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10mM glucose, and 20 mM HEPES (pH 7.4) at 37°C for 10 min.The reaction was initiated by addition of STA2. When D-609was used, it was added 10 min before addition of STA2. Afterincubation for 1–5 min, the incubation medium containing themetabolites of [3H]PC was collected in a tube, followedby centrifugation at 1,000g for 5 min. 3H radioactivity ofsupernatant (0.5 ml) was measured with a liquid scintillationcounter.

Protein assayProtein concentration was determined by the protein dye

assay (Bradford, 1976) using bovine serum albumin as a stan-dard.

Data analysisThe results were expressed as mean6 SE values. The

statistical significance of differences in the values was deter-mined by Student’st test.

RESULTS

TXA 2 receptor-mediated [Ca21]i elevation inastrocytoma cells

STA2, a TXA2 receptor agonist, increased [Ca21]i in aconcentration-dependent manner with an EC50 value of;30 nM in 1321N1 human astrocytoma cells under theculture in medium containing 5% FCS (growth medium)(Fig. 1A). In contrast, STA2 only slightly increased[Ca21]i in cells after culture for 2 days in mediumcontaining 0.5% FCS and 0.5 mM dbcAMP (differenti-ation medium) (Fig. 1A). Short-term treatment (5 min) of

FIG. 1. Effect of STA2 on Ca21 mobilization. A: Comparison ofSTA2-induced Ca21 mobilization between cells cultured in 5%FCS (growth medium; E) and 0.5% FCS containing 0.5 mMdbcAMP (differentiation medium; F). Fura 2-loaded cells werestimulated with various concentrations of STA2. Data are mean6 SE (bars) values of agonist-induced increase in [Ca21]i (n5 3–6). Resting levels of [Ca21]i were 75.9 6 3.25 (n 5 6) and87.0 6 6.28 nM (n 5 6) in cells cultured in growth medium andin differentiation medium, respectively. B: Effects of U73122 andD-609 on the Ca21 mobilization induced by STA2 in the cellscultured in 5% FCS (growth medium). Fura 2-loaded cells werepreincubated with (a) vehicle, (b) U73122, or (c) D-609 for 5 min.Then the cells were incubated with 1 mM STA2. Each tracerepresents a typical one from several experiments.


2169TXA2-INDUCED MAPK ACTIVATION

the cells with dbcAMP had no effect on Ca21 mobiliza-tion (data not shown). The STA2-induced Ca21 mobili-zation in the nondifferentiated cells was inhibited byU73122, an inhibitor of PI-PLC, whereas it was notinhibited by D-609, an inhibitor of PC-PLC (Fig. 1B).

TXA 2 receptor-mediated phosphoinositidehydrolysis in astrocytoma cells

STA2 caused accumulation of3H-labeled inositolphosphates in a concentration-dependent manner with anEC50 value of;50 nM in cells cultured in growth me-dium. The maximal accumulation of3H-inositol phos-phates was 176.36 8.9% (n5 3) of the basal value (Fig.2A). STA2 induced an accumulation of3H-inositol phos-phates to a small extent in differentiated cells, supportingthe results of Ca21 mobilization. The maximal accumu-lation of3H-inositol phosphates in the differentiated cellswas 111.66 3.9% (n5 3) of the basal value (Fig. 2A).U73122 reduced the STA2-induced accumulation of3H-inositol phosphates in the nondifferentiated cells (Fig.2B). However, D-609 had no effect on the accumulationof 3H-inositol phosphates (Fig. 2B). These resultsstrongly suggest that the STA2-induced Ca21 mobiliza-

tion in the nondifferentiated cells is the result of thephosphoinositide hydrolysis.

Effect of STA2 on MAPK activationIn cells cultured in growth medium, STA2 did not

increase or only slightly increased the phosphorylation ofMAPK analyzed by immunoblotting with phospho-MAPK antibody (Fig. 3A). In differentiated cells, how-ever, STA2 potently increased the phosphorylation ofMAPK (Fig. 3A). To examine the functional activationof MAPK, we measured the activity of MAPK as incor-poration of 32P to the substrate peptide for MAPK invitro. STA2 induced a significant MAPK activation indifferentiated cells, whereas it did induce a slight acti-vation in nondifferentiated cells (Fig. 3B). The peak ofthe activation appeared 2 min after addition of STA2 indifferentiated cells.

Effects of SQ29548, GF109203X, U73122, andD-609 on STA2-induced MAPK activation indifferentiated cells

Because STA2 caused a slight activation of phospho-inositide hydrolysis and Ca21 mobilization in differenti-ated cells, the potent stimulation of MAPK could not beexplained by a mediation of PI-PLC in the cells. Then themechanism of STA2-induced MAPK activation was fur-

FIG. 3. Activation of MAPK by STA2. A: Phosphorylation ofMAPK by STA2 in cells cultured with (a) growth medium or (b)differentiation medium. Cells were preincubated in EMEM/HEPES (pH 7.4) for 10 min before addition of the drug. Thereaction was initiated by addition of 1 mM STA2 and terminatedby addition of 0.25 ml of Laemmli’s sample buffer at the indi-cated time. The phosphorylation of MAPK was analyzed byimmunoblotting as described in Materials and Methods in detail.B: Time course of functional MAPK activation induced by STA2in nondifferentiated (E) and differentiated (F) cells. Cells wereincubated in EMEM/HEPES (pH 7.4) for 10 min before addition ofdrugs. The reaction was initiated by addition of 1 mM STA2 andterminated by addition of 0.25 ml of ice-cold lysis buffer at theindicated time. Results represent STA2-induced incorporation of32P into the substrate peptide specific for MAPK. Data are mean6 SE (bars) values (n 5 3). Asterisks indicate a significant dif-ference from control: *p , 0.05, **p , 0.01.

FIG. 2. Effect of STA2 on phosphoinositide hydrolysis. A: Com-parison of STA2-induced phosphoinositide hydrolysis betweencells cultured in 5% FCS (growth medium; E) and 0.5% FCScontaining 0.5 mM dbcAMP (differentiation medium; F). Thereaction was initiated by addition of STA2 and terminated byaddition of 1 ml of 5% trichloroacetic acid. Results were ex-pressed as percent increase of 3H-inositol phosphates (3H-IPs)from the basal value. Data are mean 6 SE (bars) values (n 5 3).B: Effects of U73122 and D-609 on phosphoinositide hydrolysisinduced by STA2 in cells cultured in 5% FCS (growth medium).The cells were preincubated in EMEM/HEPES (pH 7.4) contain-ing 10 mM LiCl for 10 min, followed by U73122 or D-609 for 10min. Then the cells were incubated with 1 mM STA2 (shadedcolumn) or vehicle (open column) for 10 min. Results representaccumulation of 3H-IPs. Data are mean 6 SE (bars) values (n5 3). Asterisks indicate a significant difference from vehicle: *p, 0.05, **p , 0.01.



ther examined in the differentiated cells. PD98059, aMEK inhibitor, significantly inhibited STA2-inducedMAPK activation, indicating the involvement of MEK inthe activation (data not shown). SQ29548, a TXA2 re-ceptor antagonist, inhibited STA2-induced MAPK acti-vation, suggesting that the activation is mediated via theTXA2 receptor (Fig. 4A).Furthermore, GF109203X, aPKC inhibitor, abolished STA2-induced activation ofMAPK, showing the involvement of PKC in STA2-induced MAPK activation. On the other hand, U73122only slightly inhibited STA2-induced MAPK activation,suggesting that PI-PLC may not be involved in theMAPK activation. Because the STA2-induced phospho-inositide hydrolysis was extremely low and the MAPKactivation was inhibited by GF109203X but was resistantto U73122 in differentiated cells, the source of diacyl-glycerol to activate PKC could be derived from a path-way other than PI-PLC.

To examine the possible involvement of PC-PLC inSTA2-induced MAPK activation, D-609 was used for the

analysis. D-609 inhibited STA2-induced MAPK activa-tion in a concentration-dependent manner with an IC50value of ;1 mM (Fig. 4B), although it did not affectphosphoinositide hydrolysis and [Ca21]i elevation in-duced by STA2 (Figs. 1B and 2B). Thus, PC-PLC maybe involved in the MAPK activation in response toSTA2.

Effect of STA2 on PC hydrolysisTo obtain direct evidence that stimulation of the TXA2

receptor induces activation of PC-PLC in dbcAMP-treated cells, the effect of STA2 on PC hydrolysis wasinvestigated. STA2 induced the release of [3H]cholinemetabolites into the medium in a time-dependent manner(Fig. 5A). Although activation of MAPK was observed 1min after addition of STA2 in dbcAMP-treated cells, therelease of [3H]choline metabolites was activated with aslight delay. This phenomenon might result from thetime lag between intracellular and extracellular elevationof levels of [3H]choline metabolites, like the case ofcarbachol-induced PC hydrolysis in 1321N1 cells (Mar-tinson et al., 1990). The release of [3H]choline metabo-lites induced by STA2 was inhibited by treatment withD-609 (Fig. 5B).

FIG. 5. Effect of STA2 on PC hydrolysis. A: Time course of PChydrolysis induced by STA2 in cells cultured with differentiationmedium. Cells were preincubated for 10 min before addition ofthe drug. The reaction was initiated by addition of 1 mM STA2,and the medium was removed at the indicated time. Resultsrepresent release of [3H]choline metabolites into the medium.Data are mean 6 SE (bars) values (n 5 3). Asterisks indicate asignificant difference from control: **p , 0.01. B: Effect of D-609on PC hydrolysis induced by STA2 in cells cultured with differ-entiation medium. The cells were preincubated for 10 min, fol-lowed by incubation with 10 mM D-609 for 10 min. Then the cellswere stimulated with 1 mM STA2 (shaded column) or vehicle(open column) for 2 min. Results represent the release of[3H]choline metabolites into the medium. Data are mean 6 SE(bars) values (n 5 3). Asterisks indicate a significant differencefrom vehicle: *p , 0.05, **p , 0.01.

FIG. 4. Effects of a TXA2 receptor antagonist and several inhib-itors on MAPK activation. A: Effects of SQ29548, GF109203X,and U73122 on functional activation of MAPK in cells culturedwith differentiation medium. The cells were preincubated inEMEM/HEPES (pH 7.4) for 10 min, followed by SQ29548 for 10min, GF109203X for 20 min, and U73122 for 10 min. Then thecells were incubated with 1 mM STA2 (shaded column) or vehicle(open column) for 2 min. Results represent incorporation of 32Pinto the substrate peptide specific for MAPK. Data are mean6 SE (bars) values (n 5 3). Asterisks indicate a significant dif-ference from vehicle: *p , 0.05, **p , 0.01. B: Effect of D-609 onMAPK activation induced by STA2. The cells were preincubatedin EMEM/HEPES (pH 7.4) for 10 min, followed by D-609 for 20min. Then the cells were incubated with 1 mM STA2 (F) or vehicle(E) for 2 min. Results represent incorporation of 32P into thesubstrate peptide specific for MAPK. Data are mean 6 SE (bars)values (n 5 3). Asterisks indicate a significant difference fromvehicle: *p , 0.05, **p , 0.01.



DISCUSSION

The present study demonstrated that stimulation of theTXA2 receptor caused MAPK activation in differentiatedastrocytoma cells treated with dbcAMP but not in non-differentiated cells. The TXA2 receptor-mediated MAPKactivation in differentiated cells is mediated through PC-PLC and PKC. It is the first demonstration that PC-PLCis involved in TXA2 receptor-mediated MAPK activa-tion.

The treatment of the cells with dbcAMP apparentlychanged the TXA2 receptor-mediated signaling from theGq–PI-PLC pathway to the PC-PLC–MAPK pathway in1321N1 human astrocytoma cells. Habib et al. (1997)reported that the human TXA2 receptor was phosphory-lated by PKC and protein kinase A (PKA). Thus, apossible interpretation of the change in signaling path-way is that dbcAMP causes the PKA-mediated phos-phorylation of the TXA2 receptor and the phosphorylatedreceptor changes its coupling ability with G protein fromGq to another G protein. On the other hand, two isoformsof the TXA2 receptor have been reported: placental type(Hirata et al., 1991) and endothelial type (Raychowdhuryet al., 1994). 1321N1 human astrocytoma cells expressboth placental and endothelial types of TXA2 receptormRNA (Honma et al., 1998). Because stimulation ofboth types of TXA2 receptor has been shown to cause[Ca21]i elevation (Yukawa et al., 1997), it is assumedthat both types are coupled with Gq. Therefore, thechange in TXA2 receptor-mediated signaling pathway isnot explained by the change in the expression ratio ofplacental and endothelial types of the TXA2 receptor.

It has been shown that TXA2 and thrombin activateG12 in human platelets (Offermanns et al., 1994; Klageset al., 1999) and that TXA2 receptor in 1321N1 humanastrocytoma cells couples with Gq and G12 (Honma et al.,1998). Recently, it has been reported that G12 activatesc-Jun NH2-terminal kinase through the monomeric Gproteins ras and rac, resulting in DNA synthesis in1321N1 human astrocytoma cells (Collins et al., 1996).Furthermore, G12 can stimulate the Na1/H1 exchangerin COS-1 cells through a PKC-dependent pathway with-out activating PI-PLC (Dhanasekaran and Dermott,1996). More recently, it has been shown that G12 stim-ulates Bruton’s tyrosine kinase and a rasGAP (Jianget al., 1998). Furthermore, it is reported that PC-PLC issignificantly relevant to cell growth (Johansen et al.,1994) and cell death (Cifone et al., 1995; Yonghonget al., 1998). Thus, it is necessary to elucidate whetherG12 is involved in the TXA2 receptor-mediated activa-tions of PC-PLC and MAPK in 1321N1 human astrocy-toma cells.

It has been reported that astrocytes (Nakahata et al.,1992) and oligodendrocytes (Blackman et al., 1998) ex-press the TXA2 receptor. However, the physiologicalrole of the TXA2 receptor in glial cells is still unknown.Astrocytes play an important role in gliosis, when theCNS is injured. Morinelli et al. (1994) reported thatTXA2 caused mitogenesis of vascular smooth muscle

cells that was mediated by activation of MAPK and S6kinase. When the blood–brain barrier was broken byposthypoxic or postanoxic injury, arachidonic acid me-tabolites and reactive lipid mediators were generated inthe brain (Bazan and Rodriguez de Turco, 1980). It hasbeen shown that cPLA2 was strongly induced in acti-vated astrocytes after transient global forebrain ischemiain the rat (Clemens et al., 1996). Furthermore, TXA2 issynthesized in glial cells by several stimuli (Ishimotoet al., 1996). We have shown in the present study thatMAPK activation induced by STA2 in differentiated cellsis greater than in nondifferentiated cells. Thus, TXA2receptor-mediated MAPK activation may represent asensitive stage in differentiated astrocytes; thus the re-ceptor may have a role in proliferation under emergencyconditions such as gliosis.

In conclusion, dbcAMP treatment of 1321N1 humanastrocytoma cells results in acceleration of the TXA2receptor-mediated activation of MAPK, in spite of thereduction of Ca21 mobilization and phosphoinositidehydrolysis. This TXA2 receptor-mediated MAPK activa-tion is mediated by PC-PLC but not PI-PLC.

Acknowledgment: The authors are grateful to Ono Pharma-ceuticals (Osaka, Japan) for the generous gift of STA2. Thiswork was supported, in part, by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science, Sports andCulture of Japan.

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Involvement of Phosphatidylcholine-Specific Phospholipase C in Thromboxane A2-Induced Activation of Mitogen-Activated Protein Kinase in Astrocytoma Cells