985Journal of Cell Science 111, 985-994 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS9701

Nuclear location of PLA 2-I in proliferative cells

Jean-Michel Fayard*, Christian Tessier, Jean-François Pageaux, Michel Lagarde and Christian Laugier

Laboratoire de Biochimie et Pharmacologie, INSERM U352, INSA de Lyon, 20 Avenue A. Einstein, B406, 69621 Villeurbanne,France*Author for correspondence

Accepted 13 January; published on WWW 9 March 1998

We have previously demonstrated that pancreatic PLA2(PLA2-I) stimulates the proliferation of UIII cells, a stromalcell line derived from normal rat uterus. In order to gainfurther insight into the mechanism of action of PLA2-I, wehave investigated the intracellular processing of PLA2-I.Either highly proliferative or growth arrested U III cellswere analyzed.Growth arrested cells were obtained from acontact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, westernblotting, immunocytochemistry and confocal microscopy,we demonstrate that endogenous PLA2-I was mainlylocated in the nucleus in highly proliferative cells whereasits location was cytoplasmic in non proliferative cells. Whennon confluent UIII cells were incubated with nanomolar

amounts of exogenous PLA2-I, the enzyme was internalizedand, in the majority of cells, appeared within the nucleus.Both internalization and nuclear location of exogenousPLA2-I were suppressed by the addition of aristolochic acidto the culture medium. Binding experiments performed onpurified nuclear preparations showed the presence ofspecific cooperative binding sites for PLA2-I. Collectivelyour data suggest that the proliferative effect exerted bypancreatic PLA2 in UIII cells is mediated by a directinteraction of the enzyme at the nuclear level. Putativemechanisms and targets are discussed.

Key words: PLA2, Proliferation, Stromal cell, Nuclear location, Ratuterus

SUMMARY

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INTRODUCTION

Phospholipases A2 (PLA2; EC 3.1.1.4) are lipolytic enzymesthat hydrolyze the sn-2 acyl ester bond in glycerophospholip(Dennis, 1994). Many forms of PLA2 enzymes have beendescribed and classified into several groups. Type I, II andPLA2 are low molecular mass (13-18 kDa), extracellulsecreted enzymes (Murakami et al., 1995), includipancreatic and cobra venom PLA2 (type I), rattle snake andinflammatory PLA2 (type II) and bee venom PLA2 (type III).Intracellular cytosolic PLA2, on the other hand, belongs to different group, including the 85 kDa (cPLA2; type IV)calcium-dependent (Clark et al., 1995) and the calciuindependent (Ackermann and Dennis, 1995) enzymes.almost every cell studied, multiple PLA2 exist, but their relativecontribution to the cell function has not yet been determin(Dennis, 1997).

The involvement of distinct types of PLA2 in cellproliferation has often been described. This is the casecPLA2, which selectively cleaves phospholipids containinarachidonic acid (AA) at the sn-2 position. Indeed, AA asprecursor of eicosanoids or more directly as a secomessenger, plays a role in gene transcription and in proliferation of various cell types (Clarke and Jump, 199The pancreatic type PLA2-I, which has long been thought toact only as a digestive enzyme, has been shown to stimucell proliferation via a specific membrane receptor (Arita et a

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1991). Cloning of PLA2 receptors from several species hasrevealed a structural organization of the protein which is highsimilar to that of mannose receptor (Lambeau et al., 199Ohara et al., 1995; Zvaritch et al., 1996). However, thphysiological role of this PLA2 receptor remains unclear(Lambeau et al., 1997).

We have recently established a stromal cell line (UIII )derived from normal rat uterus which has retained some of tcharacteristics of uterine stromal cells. Progesterone aprolactin receptor expression, progesterone regulation growth (Cohen et al., 1993) and the production oprostaglandins are hormonally regulated (Prigent et al., 199Several lines of experimental evidence demonstrated thpancreatic PLA2 plays a key function in the regulation of UIIIcell proliferation. First, the proliferation of this cell line wasreduced and even arrested by different PLA2 inhibitors but wasnot affected by inhibitors of cyclooxygenases andlipoxygenases (Fayard et al., 1994). Aristolochic acid, a PLA2inhibitor which binds directly to PLA2 and alters the α-helixcontent of the protein (Rosenthal et al., 1992), was the mopotent inhibitor with an IC50 of about 3 µM. Second, PLA2-Iadded to the culture medium of quiescent UIII cells induced asignificant proliferative effect which was dose-dependent from1 to 10 nM (Tessier et al., 1996). Third, UIII cells expressspecific membrane receptors for PLA2-I. Other secretory PLA2(Bee, Crotalus and Naja venoms PLA2), display very lowaffinity for this receptor, and correlatively showed no growth

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promoting effect (Tessier et al., 1996). Moreover, aristolocacid added to the culture medium together with pancrePLA2-I reduced the binding of the enzyme in a dose-dependmanner, with an IC50 of about 3 µM that agrees with itsantiproliferative effect (Tessier et al., 1996). Finally, we fouthat, when UIII cells progress from a non confluent proliferatinstate to a confluent contact inhibited monolayer, the numbePLA2-I receptors decreases from about 40,000 to 1,300 sper cell. These results support the conclusion that PLA2-I isinvolved in the control of UIII cell proliferation via interactionwith the plasma membrane receptor (Arita et al., 1991; Kishet al., 1992).

Time-course analysis of iodinated PLA2-I association withthe plasma membrane and the intracellular compartment ofIIIcells and cross-linking studies suggested that a single poomembrane receptors undergoes cycles of binding, intracelltransfer and release of PLA2-I followed by restoration ofbinding sites on the plasma membrane. Most of the internalenzyme was not associated with the receptor and accumuin the 14 kDa form, into the intracellular compartmenProteolysis of the internalized PLA2-I could be clearly detectedonly after 90 minutes of incubation at 37°C (Rossini et a1996). These results raise the possibility that the internalienzyme is directly responsible for the control of UIII cellfunctioning and prompted us to investigate the intracelluprocessing of the PLA2-I in proliferative and non proliferativeUIII cells. Using cellular fractionation, immunocytochemistand western blotting, we demonstrate that PLA2-I appearspreferentially to be located in the perinuclear and nuclcompartments of growing cells and in the cytoplasm quiescent cells. Moreover, confocal microscopy studshowed that, in a large proportion of growing cells, tinternalized enzyme was even located within the nucleBinding experiments on purified nuclear preparations reveathe presence of specific cooperative binding sites for PLA2-I.

MATERIALS AND METHODS

Cell cultureFor stock culture, rat uterine stromal cells (UIII cells) were grown inFalcon plastic flasks (75 cm2) in a 95% air-5% CO2 humidifiedatmosphere at 37°C. The medium was M199 supplemented with FCS, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/mlstreptomycin. Medium was changed every 48 hours. Confluent cwere subcultured by incubation with 0.25% trypsin, centrifuged aseeded at a 1:2 ratio. Cell viability, determined by the Trypan bexclusion method, was consistently greater than 95%.

Fluorescence microscopyFor immunofluorescence studies, cells were seeded (250,0050,000 cells per well for confluent and non-confluent culturrespectively) and cultivated for four days on sterile coverglasses iwell plastic culture dishes (NUNC). The culture medium was thremoved and replaced by fresh medium supplemented or not witnM PLA2-I for two hours. Cells were then rinsed twice with PBfixed in PBS-1% paraformaldehyde for 30 minutes at 0°C and wasseven times in HBSS-1% Triton X-100 for 15 minutes at rootemperature. The cells were washed four more times with HBSSBSA. Staining was performed by incubating cells overnight at 4with a polyclonal antibody against PLA2-I (final dilution 1:500) raisedin our laboratory. This antibody was specific to PLA2-I since PLA2-II was not recognized in western blotting experiments. The cro

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reaction analyzed by dot blots was less than 0.1% (Prigent-Tessieal., 1996). The staining was followed by four washes with HBSS-1BSA. The cells were then exposed for 30 minutes at 4°C to tsecondary fluorescent TRITC-conjugated antibody in HBSS-1% BSand washed four times in the same solution without the antibody. Tcoverglasses were detached from the culture wells, placed on a gslide and coverslipped using PBS-glycerol (50-50) as mountisolution. Controls were treated with preimmune rabbit serum. The cpreparations were analyzed using a fluorescence microscope (LLaborlux) or a confocal microscope (Zeiss, LSM-10, OberkosheGermany).

Western blotting analysisUIII cells cultivated in 60 mm diameter Petri dishes were trypsinizand centrifuged. The pellet was washed twice with PBS without Ca2+

and Mg2+ and resuspended in RIPA buffer (150 mM NaCl, 0.1%Nonidet P40, v/v, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCpH 8.0) with 1 mM PMSF, 2 µg/ml leupeptin and aprotinin. Aftersonication (20 seconds in ice), the lysate was aliquoted and store−20°C.

The protein content of the lysate was determined using the micBCA (bicinchoninic acid) assay from Pierce (Rockford, USAaccording to the manufacturer’s indications. The DNA content wdetermined by a fluorometric procedure derived from the techniqof Labarca and Paigen (1980) as previously described (Tessier et1996).

Aliquots of the lysates containing the same quantity of eithprotein (40 µg) or DNA (2 µg) were diluted twice in 2× Laemmlibuffer, 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, v/v0.002% Bromophenol Blue. Protein denaturation was obtained heating at 100°C for 5 minutes and disulfide bonds were reduced5% β-mercaptoethanol. Samples were then submitted to SDS-PAunder reducing conditions according to the method of Laemmli (197by using a 3% acrylamide stacking gel (30% acrylamide/biacrylamide, 125 mM Tris-HCl, 0.1% SDS, pH 6.8, water 0.5/2.5/v/v/v) and a 12% acrylamide separation gel (30% acrylamide/bacrylamide, 375 mM Tris-HCl, 0.1% SDS, pH 8.8, water 8/10/2v/v/v).

Electrophoresis was for about 2 hours in electrophoresis buffer (mM Tris-HCl, 192 mM glycine, 0.1% SDS, pH 8.3) at constancurrent (25 mA/gel) in a refrigerated vertical tank (Mighty small ISE 250 Hœffer Scientific Instrument). Proteins were theelectrotransferred to nitrocellulose membrane (0.2 µm, Schleicher andSchuell) in a semi liquid system (Mini trans blot Bio-Rad), at constavoltage (100 V) for 1 hour, in a cold transfer buffer (25 mM Tris-HC192 mM glycine, 20% methanol, pH 8.3).

Immunodetection was performed in cold saline buffer (20 mM TriHCl, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBS-T). Thnitrocellulose membrane was dried, then incubated overnight in TBT containing 5% non fat dry milk. The incubation with the primarantibody against PLA2-I, raised in our laboratory (Prigent-Tessier eal., 1996) and used at a final concentration of 1/5,000, lasted fohours. The incubation with the secondary antibody, conjugatedhorseradish peroxidase (dilution 1/5,000), lasted for 1 hour. Proteantibody complexes were visualized using the enhancchemiluminescence (ECL) detection system (Amersham, Les UFrance). The band densities were determined by scanndensitometric analysis using the bioprofil scanner (Vilbert-LourmaMarnes la Vallée, France).

Assay of PLA 2 activitySecreted PLA2 activity was determined by using a fluorometricmethod derived from the technique of Radvanyi et al. (1989). UIII cellscultured in 60 mm diameter Petri dishes were trypsinized, washtwice in 2 ml PBS without Ca2+ and Mg2+ and resuspended in anappropriate volume of this buffer. Then the cells were sonicated 15 seconds at 0°C and the lysates were stored at −20°C. For PLA2

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activity measurement, 100 µl of 2 mM PG substrate (1-hexadecanoy2-(10-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammoniumInterchim, Montluçon, France) stored at −80°C in toluene-isopropanol(50/50, v/v) were evaporated under nitrogen and dissolved in 1ethanol. 100 µl of this solution were mixed with 10 ml of 50 mM Tris-HCl buffer, 100 mM NaCl, 1 mM EGTA, pH 7.5, vortexed for 2minutes and maintained at room temperature.

Incubation medium was prepared in a fluorometric cuvette adding successively 1,940 µl of the PG substrate (2 µM finalconcentration) and 20 µl of fatty acid free BSA solution (0.1% finalconcentration). 10 to 50 µl aliquots of lysates kept at 0°C were addeand equilibrated at room temperature for 1 minute. The reaction started by adding 20 µl of 1 M CaCl2 and fluorescence was measurecontinuously on a spectrofluorometer Jobin Yvon JY3D. SecrePLA2 activity was estimated by measuring the slope of the curve aexpressed as pmoles of substrate hydrolyzed/minute per mg DNA

Isolation of nucleiCell homogenisation procedures that use ionic salt solutions wavoided since they often induce artefactual transfer of molecules fthe nuclear compartment to the cytoplasm. Nuclei were prepaaccording to the method of Nicotera et al. (1989) with sommodifications. UIII cells from subconfluent stock cultures wertrypsinized and centrifuged at 800 g for 5 minutes. The pellet wasrinsed twice with PBS and resuspended into three volumes of TKsolution (50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 2µg/ml leupeptin and pepstatin with 0.1% Triton X-100) supplementwith 0.25 M sucrose, briefly sonicated using a microprobe acentrifuged again at 700 g for 10 minutes at 4°C. The resulting pellewas resuspended into 2 vols of the above TKM-0.25 M sucrosolution, then 1 vol. of sample was gently mixed with 2 vols of TKsupplemented with 2.3 M sucrose. TKM-2.3 M sucrose (1 vol.) wplaced into centrifuge tubes (ultraclear no. 344057, Beckman) andvols of experimental sample were layered on the top. The tube centrifuged at 37,000 g for 45 minutes at 4°C. The supernatant waaspirated and the nuclear pellet was resuspended in TKM solutionpelleted at 1,000 g for 5 minutes at 4°C. The final nuclear pellet waresuspended in 50 mM Tris-HCl, pH 7.5, and analyzed. The DNcontent of the sample was determined in an aliquot by the fluoromeprocedure described above. For electron microscopy, samples wused immediately. For binding studies, samples were stored at −80°Cuntil use.

Electron microscopyBlocks of pelleted nuclei were fixed in 1% paraformaldehyde in 0M cacodylate buffer for 2 hours, post-fixed with 1% OsO4 and thendehydrated. Ultrathin sections were cut, stained with uranyl aceand examined with a Philipps CM120 electron microscope. For esample, several sections were observed from different regions ofblocks, in order to ensure sufficient purity of the nuclear preparatioand the absence of membrane contamination. Data presented in5 are representative of the range of samples observed.

Binding studiesAliquots of the nuclei preparations (100 µl) were incubated with theindicated concentrations of iodinated PLA2-I in the presence orabsence of a 100-fold molar excess of non-radioactive PLA2-I as acompetitor. Iodination of porcine pancreatic PLA2 was carried outusing the iodo beads iodinating reagent, as decribed previou(Rossini et al., 1996). The binding medium consisted of M1containing 0.1% (w/v) BSA and 50 mM Hepes buffer, pH 7.2. Thtotal volume of incubation was 250 µl containing 15 to 50µg DNA.The incubation with the ligand was performed for 15 minutes at 37then each sample was filtered through a 2.5 cm diameter gmicrofibre filter (GF/B, Whatman) under vacuum. The filter was thwashed five times with 5 ml of ice cold PBS and counted by liquscintillation using 5 ml of Ultimagold (Packard). The specific bindin

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of PLA2 to nuclei was determined by subtraction of the radioactividetected in the samples incubated in the presence of competitor (specific binding) from that of paired samples incubated in its abse(total binding). Analysis of total, non specific and specific binding wperformed using JMP software (SAS institute) and weighted nlinear regression. The maximal binding values were expressedpmol/mgDNA rather than pmol/mg of total protein, the conventionunit, since binding experiments were performed on purified nuclepreparations. Preliminary time course assays have shown that at 3the specific binding reached a plateau after 10 minutes and remaat the maximum level for about 40 minutes, thus a 15 minuincubation time was chosen for standard experiments.

Statistical analysisData are expressed as means ± s.e.m. Statistical significance (α=0.05)was tested using Mann-Whitney or Kruskal-Wallis non parametrrank tests (Conover, 1980).

RESULTS

Activity and subcellular distribution of PLA 2-I inproliferative and non proliferative U III cells Secreted PLA2 activity, measured in cell lysate was greatlreduced (−66%, P<0.05) when UIII cells progressed from anactively proliferating state to a contact inhibited monolay(Fig. 1a). Western blotting analysis after SDS-PAGE showthat the intracellular concentration of PLA2-I was significantlyreduced, about threefold, in confluent cells (Fig. 1b,c), decrease which is in accordance with the variation of PL2activity. UIII cells also express an immunoreactive PLA2-II,detected by western blotting with a polyclonal antibody raisin our laboratory against type II PLA2 (obtained from thevenom ofCrotalus atrox). In contrast to what we observed withPLA2-I, PLA2-II was present at the same concentration confluent and in non-confluent cells (data not shownImmunofluorescence microscopy studies (Fig. 2) show that staining pattern was very dependent on the proliferative stof the cells: confluent cells expressed a low level of PLA2-I(Fig. 2c) and the enzyme was distributed throughout tcytoplasm. On the contrary, the expression of PLA2-I washighly enhanced in non-confluent cells (Fig. 2g) and thstaining was mainly located in the perinuclear and nuclecompartments, which suggests a high PLA2-I concentrationaround and within the nucleus of growing cells. Westeblotting analysis of the subcellular distribution of the enzymin confluent and non confluent UIII cells confirms these results(Fig. 3). PLA2-I was hardly detectable in the soluble fractioof confluent and non confluent cells (Fig. 3a, lanes 1-2) awas found at almost the same concentration in the particufraction of both groups of cells (Fig. 3a, lanes 3-4). Aexpected, a large difference was apparent in the nuclear conof the enzyme between the two groups of cells (Fig. 3a, lan5-6), PLA2-I was about 3.5-fold more concentrated in thnuclear fraction of non confluent cells.

Nuclear localization of exogenous internalized PLA 2-IUIII cells express specific membrane receptors for PLA2-Iwhich undergo cycles of binding, intracellular transfer anrelease of the enzyme (Rossini et al., 1996). As in oexperimental model, the internalized enzyme is not rapiddegraded, we undertook experiments to study its intracellu

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Fig. 1. Secreted PLA2 activity and PLA2-I expression in nonconfluent and confluent UIII cells. Non confluent UIII cells (3 daysafter plating) were harvested for subsequent measurement of PLA2activity and PLA2-I expression as described in Materials andMethods. Confluent UIII cells (7 days after plating) from the samestock culture were harvested and processed in the same way. PL2activity (a) is expressed as nanomoles of substrate hydrolyzed peminute and per mg DNA. Each group includes measurements mafrom three independent experiments (mean ± s.e.m.). ExpressionPLA2-I was measured by western blotting as described in Materiaand Methods. (b) A characteristic immunoblot (lane 1 correspond50 ng porcine pancreatic PLA2), and (c) the data obtained afterquantification of the bands using a videodensitometer. Results areexpressed as arbitrary units of optical density, the values obtainedconfluent cells were arbitrarily fixed at 100%. Data are the mean ±s.e.m. of 8 independent experiments. Intracellular concentrations,calculated from a standard curve are 4.38±0.67 and 1.33±0.21 ngµgDNA for non confluent and confluent cells, respectively.

localization. To this end, proliferative and non proliferativUIII cells were preincubated for 90 minutes with 20 npancreatic porcine PLA2 and the intracellular localization othe internalized enzyme was studied bimmunocytochemistry. Two kinds of non proliferative celwere used, either contact-inhibited confluent monolayersnon confluent cultures pretreated with 3µM aristolochic acid(Fayard et al., 1994). Results presented in Fig. 2 show the fluorescent labeling increases a lot in growing ceincubated with PLA2-I compared to control (Fig. 2h versug) and the fluorescence appears mainly associated withnucleus. The cellular staining was significantly reduced whnon-confluent cells were incubated in the presence aristolochic acid (Fig. 2l versus h). These results confirm internalization of the exogenous PLA2-I. The processappeared much more important in growing (Fig. 2h versusthan in quiescent confluent cells (Fig. 2d versus c) whichconsistent with the decrease in the number of PLA2-Imembrane receptors observed previously in UIII cells as theyprogress from an actively proliferating state to a conflue

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state (Tessier et al., 1996). It should be noted that when nconfluent UIII cells were incubated in the absence oexogenous PLA2-I but in the presence of aristolochic acid,the fluorescent labeling decreased, particularly in the nuclecompartment (Fig. 2k versus g). This result suggests thendogenous PLA2-I was secreted, then internalized via themembrane receptor and finally migrated to the nucleus. growing cells, two characteristic patterns of fluorescencstaining were observed. In some cells, the fluorescence wessentially located around the nucleus, in a region whicseems to correspond to a particular cytoplasmic organizatioIn the majority of cells (about 65%), the fluorescence walocated in the nucleus. In order to get a more precisintracellular localization of the enzyme, we finally performedconfocal microscopic studies. Fig. 4 shows optical sectionin the z plane of a cell showing a typical nuclear staining. Thfluorescence clearly appeared located within the nucleuNeither the nuclear envelope, nor the cytoplasm wersignificantly stained.

UIII cells possess nuclear bindings sites for PLA 2-ITo gain more information on the mechanism responsible fothe nuclear location of PLA2-I, we performed bindingexperiments on nuclear preparations. As shown by electrmicroscopy, the experimental procedure used allowed us obtain highly pure preparations of nuclei, without nucleaenvelope and with no evidence of substantial amounts of extrnuclear debris (Fig. 5). When nuclear preparations of UIII cellswere incubated with increasing concentrations of iodinatePLA2-I, elevated levels of non specific binding increasinglinearly with ligand concentrations were observedNevertheless, comparison of total to non specific bindindemonstrated the presence of a low but saturable level specific binding, reaching an apparent plateau foconcentrations of ligand exceding 10 nM (Fig. 6a). Scatcharepresentation of the data from this experiment showed that tbinding does not follow the simple Michaelis Menten equatio(Fig. 6b). On the contrary, when the data were plotteaccording to the Hill representation, a good fit was observe(Fig. 6c).

Weighted regression analysis confirmed the good agreemeto the Hill model (R2=0.93) and the values of the twocharacteristic coefficients were of K′=257±92 andn=2.23±0.33 with a maximal binding value of Bmax=0.39±0.06pmol/mg DNA. Two other independent experiments, realizewith two other nuclear preparations, showed similar bindinprofiles to those reported in Fig. 6a, but with different Bmaxvalues: 0.28±0.05 and 0.42±0.08 pmol/mg DNA. Thisvariability in the maximal binding capacity of different nuclearpreparations is not surprising and has to be related to tvariability of biological preparations. On the contrary, the Hilcoefficients were very close, which confirms the potential othis model to describe our experimental data. Thus, in orderdefine more accurately these parameters, we analyzsimultaneously the three sets of experiments using a Hill modin which K′ and n were independent of the set and Bmax wascharacteristic of each experiment. The estimated values of tHill parameters obtained (from 32 experimental points) bweighted non linear regression were: K′=305±186,n=2.47±0.31 and Bmax comprised between 0.24±0.05 and0.58±0.07 pmol/mgDNA.

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Internalized PLA 2-I is located in the nucleus ofproliferative U III cellsPLA2-I expression in UIII cells varied both quantitatively andqualitatively according to the proliferative state of the celThe intracellular concentration of PLA2-I was significantlyreduced in confluent cells, and this reduction was closassociated with the reduction in enzyme activity. Thedifferences only come from changes in the concentrationPLA2-I in the nuclear fraction. Moreover, using varioumethods, we confirmed that in growing cells exogenous PL2-I was internalized and we demonstrated that in the majoritycells the enzyme migrated into the nucleus, where it coulddirectly active at the nucleoplasmic level.

Our findings on accumulation of PLA2-I in UIII cells are in

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line with the proposal that membrane receptors foextracellular secreted PLA2 contribute to clearance of thecirculating enzyme (Ancian et al., 1995). Intracellularlocalization of PLA2-I has been previously described in normaand ras-transformed rat kidney cells (Bar-Sagi et al., 198where the enzyme appeared diffusely distributed throughothe cell. Our observation of two main characteristic staininpatterns in growing and growth arrested cells probabindicates that the level of the internalized enzyme does not onreflect an equilibrium between the extracellular anintracellular compartments, but rather a specificphysiologically relevant mechanism depending on thphysiological status of the cell, perhaps in relation to the cecycle. However, the molecular mechanism(s) underlying thaccess of PLA2-I to the cytosolic, and even to the nuclearcompartments, is (are) presently unknown. The classic

Fig. 2. Immunofluorescencedetection of PLA2-I in confluent andnon confluent UIII cells. Confluent(a-d) and non confluent (e-l) UIIIcells (4 days after plating) wereincubated for 90 minutes with(a,b,d,e,f,h,i,j,l) or without (c,g,k)20 nM porcine pancreatic PLA2 inthe presence (i-l) or absence (a-h) of3 µM aristolochic acid. Cells weretreated for immunocytochemistry asdescribed in Materials and Methods.Fixed cells were permeabilized andincubated without serum (a,e,i),with normal rabbit serum (b,f,j) orpolyclonal antibody against PLA2-I(c,d,g,h,k,l), followed by TRITC-conjugated antibody against rabbitIgG and observed using afluorescence microscope. Bar, 5 µm.

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Fig. 3.Subcellular distribution of PLA2-I in confluent and nonconfluent UIII cells. Confluent (C, lanes 2, 4, 6) and non confluent(NC, lanes 1, 3, 5) UIII cells were trypsinized, resuspended in PBSwithout Ca2+ and Mg2+ and sonicated as described in Materials andMethods. Sonicates were centrifuged (1,000 g, 10 minutes, 4°C) andthe resulting supernatants were centrifuged again (108,000 g, 60minutes, 4°C). The 108,000 g supernatants (referred to as cytosol,lanes 1 and 2), the corresponding pellets (referred to as particulatefraction, lanes 3 and 4) and the 1,000 g pellets (referred to as nuclearfraction, lanes 5 and 6) were analyzed by SDS-PAGE (fractions from1.25×106 cells/well, except lanes 5 and 6 which contain fractionsfrom 3.75×106 cells/well), and immunoblotting for PLA2-I. The datain a are from one representative experiment out of three. The bandswere quantified using a videodensitometer and the PLA2-Iconcentration in the particulate and nuclear fractions, expressed asarbitrary units of optical density, is reported in b. Results are themean ± s.e.m. of three independent experiments.

Fig. 4.Confocal fluorescence microscopy study on the nuclearlocalization of internalized PLA2-I. Non confluent UIII cells wereincubated for 90 minutes with 20 nM porcine pancreatic PLA2 andthen treated for indirect immunofluorescence detection of PLA2-I asdescribed in Materials and Methods. Stained cells were observedwith a confocal microscope, 15 optical sections in the zaxis wereobtained by scanning from the apical surface of the cell to theadherent base in 0.5 µm step sizes. Seven characteristic micrographsof a cell showing a typical nuclear staining pattern are presented.Left panels: phase contrast controls. Right panels: confocalmicrographs revealing an intense nuclear staining, no substantialstaining was observed in cytoplasm.

mechanism which implies a specific proteolytic cleavage, donot necessary exclude the possibility that the product of tcleavage again reacts with antibodies against the inimolecule. Previous works (Arita et al., 1991; Ohara et a1995) have demonstrated that after binding to its receptor, hydrolytic activity of PLA2-I was greatly reduced. Structuradata (Van den Berg et al., 1995) indicate that in solution PLA2-I molecules show important disorders for the N-terminal regiwhich could be related to the low activity of the enzyme. Thuwe may imagine a release of the internalized PLA2-I from theendosomal vesicles (for example after acidification of tmedium) into the cytosol and its transfer toward the nuclethe low molecular mass of PLA2-I making possible theimportation of the enzyme into the nucleus via the nuclepores.

The nucleus is now considered a highly organized organein which specific functions are located in specific nuclecompartments. For instance, phosphoinositide-5 kina

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diacylglycerol kinase and phospholipase C have been localiin the internal nuclear matrix while phosphoinositide-4 kinawas found to be associated with the peripheral matstructures (Payrastre et al., 1992). The role of variophospholipases A2 in the production of intracellular signals athe plasma membrane has been well documented. In contlittle is known about their role in other cellular compartmenHowever, there is emerging evidence that these enzymesalso involved in the production of signals within the nucle(Divecha et al., 1997). Our data on the nuclear location of

Fig. 5.Electron microscopy of nuclearpreparations. Nuclear preparations from UIIIcells obtained as described in Materials andMethods were immediately fixed inglutaraldehyde (1% final), sodium cacodylatebuffer (0.1 M final), post-fixed in 1% OsO4,embedded in Araldite and thin sections wereobserved with a electron microscope (PhilippsCM120) to check the purity of the preparation.(a) Isolated nuclei at low magnification (bar, 1.5µm). Note the presence of only a small amountof extranuclear debris. (b) Higher magnification(bar, 0.5 µm). Both dispersed and condensedchromatin domains are visible. Note theabsence of nuclear membrane.

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PLA2-I are the first description of such intracellularlocalization for pancreatic PLA2, but nuclear location of otherenzymes implicated in the metabolism of arachidonic acid halready been shown. Particularly, the subcellular location of t85 kDa, cytosolic PLA2 (cPLA2), has been recentlyinvestigated in endothelial cells at various proliferative stag(Sierra-Honigmann et al., 1996). In tightly confluent cells, thcPLA2 is located in the cytoplasm whereas in subconfluecells, the enzyme was essentially found at the nuclear levThe nuclear translocation of cPLA2 in response to various

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Fig. 6. Equilibrium binding of 125I-PLA2-I topurified nuclei from UIII cells. Nuclei from nonconfluent UIII cells were prepared as described inMaterials and Methods. The nuclei were incubatedfor 15 minutes at 37°C with 125I-PLA2 in thepresence (non specific) or absence (total) of a 100-fold molar excess of cold competitor. The specificbinding was calculated by subtracting the nonspecific from the total binding. Each valuecorresponds to the mean of 2 or 3 independentdeterminations. (a) Saturation curves of 125I-PLA2binding representative of one from threeindependent experiments. The specific binding dataobtained in this experiment are plotted according tothe Scatchard (a) or the Hill (c) representation whereBmax represents the maximum binding estimatedgraphically from a.

stimuli has been described in various models (Peters-Goland McNish, 1993; Glover et al., 1995; Schievella et al., 19Peters-Golden et al., 1996). Moreover, cyclooxygenase-2 5-lipoxygenase, two enzymes downstream in the arachidoacid cascade, have also been shown to associate withnucleus in stimulated cells (Morita et al., 1995; Schievellaal., 1995). The colocation of cPLA2, 5-lipoxygenase and 5-lipoxygenase-activating protein at the nuclear membrane also been recently described (Pouliot et al., 1996). Tnoteworthy nuclear location concerns enzymes implicatedthe arachidonic acid metabolism that are known to translocated to membranes by calcium influx (Murakami et 1995). For PLA2-I functioning, calcium only plays a role inpositioning the substrate. Thus, the nuclear location of PLA2-I results from a different mechanism and binding experimeon nuclear preparations from non confluent cells suggestpresence of nuclear binding sites for PLA2-I.

Proliferative U III cells possess nuclear binding sitesDespite a high level of non specific binding, bindinexperiments demonstrate the presence of a low but saturspecific binding of PLA2-I to purified nuclei. These data cannobe analyzed assuming a simple Michaelis-Menten model more probably they reveal cooperative properties. It shouldnoted that elevated values of non specific binding mintroduce a great imprecision in the determination of specbinding, and complementary experiments are necessaryconfirm the existence of nuclear binding sites or nuclear targfor PLA2-I. Nevertheless we may speculate that these binddata are physiologically relevant, since PLA2-I is observedwithin the nucleus only when the intracellular concentrationthe enzyme is high. The presence of specific saturable bindsites does not necessary imply the presence of a spereceptor, but may also indicate molecular interactions betw

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PLA2-I and other components of the nuclear matrix. ThuPLA2-I might appear with ambivalent functions: an enzymepotentially interacting with phospholipid substrates, and ligand, binding to a still unknown molecule. Cooperativebinding data could result from an interaction between thethree components.

What role does PLA 2-I play in the nucleus?Since we bring evidence for the nuclear location of PLA2-I ingrowing UIII cells, an important question which has to beanswered is: how does PLA2-I act in the nucleus?

One possibility is that the internalized PLA2-I exerts itshydrolytic activity within the nuclear compartment and, thusmakes possible a direct action of specific fatty acidparticularly arachidonic acid, which is now considered as new intracellular second messenger which can be involvedthe regulation of cell growth and proliferation by a direcgenomic action (Graber et al., 1994). An arachidonic acresponse element has even been recently identified (Clarke Jump, 1996). The presence inside the nucleus of lipids thcould be potential substrates for secreted PLA2 was reportedlong ago (Gurr et al., 1963; Rose and Frenster, 1965) but hnot received much attention. Initially considered as structurelements (Manzoli et al., 1972) these nuclear phospholipimay also be involved in the control of genomic activities asuggested by their higher concentration in active chromat(Frenster, 1969). Changes in fatty acid composition of nuclephospholipids have also been described during liveregeneration after partial hepatectomy (Ishihara et al., 199These changes have been associated with a neutral PL2activity identified in the nuclear matrix (Tamiya-Koizumi et al.1989). The presence of a specific PLA2 activity in the rat livernuclear matrix, which differs from that of other cellularcompartments, has been recently confirmed (Neitcheva a

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Peeva, 1995). Furthermore, crosstalk between PLA2-I andother types of PLA2 cannot be excluded since a number of dasupport such interactions. First, in rat mesangial ceinternalization of PLA2-I increases the expression and thactivity of a PLA2-II (Kishino et al., 1994). However, becauswe observed the same expression of PLA2-II in confluent andnon confluent UIII cells, it is unlikely that PLA2-I controlsPLA2-II expression and activity in UIII cells. Second,incubation of 3T3 fibroblasts with exogenous PLA2-I for morethan 6 hours significantly increased the release of arachidacid (Xing et al., 1995), the maximal effect being observafter 40 hours of incubation. The authors demonstrated thatactivation did not result from the enzymatic activity of PLA2-I but rather from the activity of a Ca2+-independent PLA2. Itis worth noting that UIII cells possess a Ca2+-independent PLA2which is more active in non confluent than in confluent ce(unpublished data). Thus, there is increasing evidence that PLA2 exerts a specific role in cell activation and signtransduction (Balsinde and Dennis, 1996).

Another possibility is that internalized PLA2-I exertsphysiological effects independently of its hydrolytic activitand acts as a cytokine or a growth factor via a direct actiothe nuclear level. The nuclear target of the PLA2-I detected inthis study could be one of the members of the nuclear recegroups. These molecules include the steroid hormone recefamily which have two important regions: one forms two ‘zinfingers’ and is responsible for the interaction with DNA, thsecond is the ligand-binding domain (Evans and Hollenbe1988). Moreover, previous studies have revealed the preseof other domains which are the sites of additional functiosuch as protein-protein interactions that participate transcriptional regulation (Yang et al., 1990). Works are progress to test this hypothesis.

Electron microscope studies were carried out at the CMEABLyon I and confocal microscope studies at the ‘Centre CommunQuantimétrie-Lyon I’. We thank A. Rivoire, C. Souchier and CNardon for skilful technical assistance and H. Cohen for providing UIII cells used in this study. This work was supported by INSERand C.T. was a recipient of a MER grant fellowship. We are grateto V. Pommatau-Deschamps for her excellent secretarial assistan

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