Involvement of Phosphatidate Phosphohydrolase in Arachidonic Acid Mobilization in Human Amnionic WISH Cells*

María A. Balboa, Jesús Balsinde, and Edward A. DennisDagger

From the Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California at San Diego, La Jolla, California 92093-0601

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Prostaglandins are known to play a central role in the initiation of labor in humans, and amnionic cells constitute a major source of these compounds. Prostaglandin synthesis and release by amnion cells in response to hormones and ligands takes place after a characteristic 4-5 h lag. However, we report herein that free arachidonic acid (AA), the metabolic precursor of prostaglandins, can be induced at much shorter times (1 h) in human amnionic WISH cells by phorbol 12-myristate 13-acetate (PMA) through activation of protein kinase Calpha (PKCalpha ). WISH cells were found to possess both cytosolic group IV phospholipase A2 (cPLA2) and Group VI Ca2+-independent phospholipase A2 (iPLA2). Of these, the cPLA2 was found to be the likely mediator of AA mobilization in PMA-activated WISH cells. PMA also activates phospholipase D (PLD) in these cells and ethanol, a compound that inhibits PLD-mediated phosphatidic acid (PA) formation, blocked AA release. Moreover, prevention of PA dephosphorylation by the PA phosphohydrolase inhibitors propranolol and bromoenol lactone, resulted in inhibition of AA release by PMA-treated WISH cells. Collectively, these data suggest that activation of cPLA2 and attendant AA release by phorbol esters in WISH cells requires prior generation of DAG by phosphatidate phosphohydrolase.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phospholipase A2 (PLA2)1 constitutes a key regulatory step in the production of prostaglandins (PGs) because it catalyzes the release of arachidonic acid (AA) from the sn-2 position of phospholipids, making the fatty acid accessible to PG synthases. At present, ten different PLA2 groups have been identified (1-3). Those include five groups of small secreted PLA2s, which show millimolar requirements for Ca2+ (Groups I, II, III, V, and X), and two groups of intracellular, high molecular weight enzymes (Groups IV and VI). Group IV PLA2, or cPLA2, is Ca2+-dependent and a highly regulated enzyme (4); whereas, Group VI PLA2, or iPLA2, is Ca2+-independent (5). At present it is not known whether Group VI iPLA2 is subjected to post-translational regulation (5). Among these PLA2s, Groups II, V, and IV have been shown to be the responsible enzymes for prostaglandin generation in different systems (6-8). On the other hand, Group VI PLA2 has been implicated in basal fatty acid remodeling reactions (5, 9).

PGs, especially PGE2 and PGF2alpha , are thought to play a central role in the initiation of spantaneous labor in humans by mediating physiological effects such as uterine contractions (10) and cervical softening and effacement (11). The human amnion has the capacity of producing PGE2, and it is known that changes in this capacity occur in association with parturition (12). Thus, numerous studies have focused on PG production by amnionic cells, mostly at the level of PG synthase enzymes (13-15). Surprisingly however, the study of PLA2 in amnion cells has received much less attention. Myatt and co-workers (16, 17) recently documented the enhancement of cPLA2 protein by interleukin (IL)-1beta in amnionic WISH cells after an 8-h treatment, which correlates with PGE2 production under those conditions. These studies were conducted at late stages of activation (several hours). Unfortunately, no information is available on the events that occur immediately after amnionic cell activation (i.e. up to 1 h). In the current study, we have investigated the signaling mechanisms that operate at the early stages of WISH cell activation and lead to increased PLA2 activity and concomitant AA release.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials-- Human WISH cells (established amnion cell line) were obtained from the American Type Culture Collection (Rockville, MD). Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was from BioWhittaker (Walkersville, MD). Fetal bovine serum was from Hyclone Labs. (Logan, UT). Trypsin/EDTA solution was purchased from Irvine Scientific (Santa Ana, CA). (5,6,8,9,11,12,1,15-3H)Arachidonic acid (specific activity 100 Ci/mmol), (9,10-3H)palmitic acid (specific activity 43.3 Ci/mmol), and 1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phosphocholine (specific activity 55 mCi/mmol) were obtained from NEN Life Science Products (Boston, MA). 1-Palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine (specific activity 59 mCi/mmol). Bromoenol lactone (BEL) was from Biomol (Plymouth Meeting, PA). Group VI iPLA2 antiserum was generously provided by Dr. Simon Jones (Genetics Institute, Cambridge, MA). Group IV cPLA2 antibodies were kindly provided by Dr. Ruth Kramer (Lilly Research Laboratories, Indianapolis, IN). The sPLA2 inhibitor LY311727 was kindly provided by Dr. Edward Mihelich (Lilly Research Laboratories). Rabbit polyclonal anti-ERK-2 that recognizes p42 and p44 MAPKs were a generous gift from Dr. Alan Saltiel (Parke-Davis, Ann Arbor, MI). Methyl arachidonyl fluorophosphonate (MAFP) was from Cayman (Ann Arbor, MI). Antibodies against PKCalpha , PKCbeta , and PKCepsilon and the polyconal anti-Raf-1 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against PKCgamma , PKCdelta , and PKCzeta were purchased from Calbiochem.

Cell Culture-- WISH cells (18) were maintained in Iscove's modified Dulbecco's medium suplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere at 90% air and 10% CO2. The cells were subcultured twice weekly by trypsinization and, when used for experiments, were seeded into 24-well (2 × 105 cells/well, NUNC) or 12-well plates (5 × 105 cells/well, Corning Inc.), or 100 × 20-mm dishes (2.5 × 106 cells/dish, Falcon). After a 2-day growth, the cells, at 90% confluency, were rinsed with serum-free medium and incubated for 1-2 h before stimulation.

PLA2 Assays-- Ca2+-dependent PLA2 assay was conducted as described by Kramer et al. (20), with slight modifications. Briefly, aliquots of WISH cell homogenates were incubated for 30 min at 37 °C in 2 mM CaCl2, 50 mM Hepes, pH 7.4, and sonicated liposomes consisting of 2 µM 1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phosphocholine and 1 µM 1,2-dioleoyl-sn-glycerol. Products were analyzed by thin-layer chromatography using the system n-hexane/diethyl ether/acetic acid (70:30:1). To measure Ca2+-independent PLA2 activity, aliquots of WISH cell homogenates were incubated for 30 min at 37 °C in 100 mM Hepes, pH 7.5, 5 mM EDTA, 0.8 mM ATP, 400 µM Triton X-100, 100 mM 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine, in a final volume of 500 µl. The substrate was used in the form of mixed micelles of Triton X-100/phospholipid at a molar ratio 4:1, obtained by a combination of heating, vortex mixing, and water bath sonication (21). Products were analyzed by thin-layer chtomatography using the same system described above.

[3H]AA Release-- Radiolabeling of the cells with [3H]AA was achieved by including 0.5 µCi [3H]/106 cells in the culture medium 20 h before stimulation. Cells were stimulated with PMA (25-50 ng/ml) for different periods of time in the presence of 1 mg/ml bovine serum albumin (fatty acid-free). The supernatants were removed and cleared of detached cells by centrifugation, and radioactivity was counted by liquid scintillation. When inhibitors were used, they were added to the cells 30 min before PMA was added to the medium.

PKC Activity-- A Promega kit (PKC assay system, V5910) was used for this purpose, and the manufacturer instructions were followed. Briefly, the cells were washed with phosphate-buffered saline, resuspended in 0.5 ml of extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM beta -mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.5 mM PMSF) at 4 °C, and homogenized using a Dounce homogenizer. Lysates were centrifuged in a microcentrifuge for 5 min at 4 °C, and supernatants were passed through a 1-ml column of DEAE cellulose pre-equilibrated with the extraction buffer. PKC was extracted by using the extraction buffer plus 200 mM NaCl. PKC activity was then measured with a biotinylated peptide substrate of PKC that binds to Streptavidin-disks. PKC was assayed in 25 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.3 mg/ml phosphatidylserine, 30 µg/ml DAG, 25 µM EGTA, 400 µM CaCl2, and [gamma -32P]ATP (5000 pmol, 100-200 cpm/pmol). Reactions were run with and without phospholipids and stopped by adding 7.5 M guanidine-HCl. Aliquots from the reactions were spotted in streptavidin-disks and washed with 1 M NaCl, and radioactivity was quantified by scintillation counting.

PKC Translocation Assays-- Experiments were carried out as described elsewhere (20). Briefly, cells were plated in 100-mm dishes. Control and PMA-stimulated WISH cells were washed with phosphate-buffered saline and homogenized with a Dounce homogenizer in a buffer consisting of 20 mM Tris-HCl, 2 mM EDTA, 10 mM EGTA, 1 mM PMSF, 20 µM leupeptin, 20 µM aprotinin, and 0.1% beta -mercaptoethanol, pH 7.5. Homogenates were centrifuged at 500 × g for 5 min at 4 °C. The resulting supernatant was centrifuged at 100,000 × g for 1 h at 4 °C to separate soluble and membrane fractions. Membranes were washed with buffer, resuspended, and sonicated. After protein quantification, 100 µg were separated by SDS-polyacrylamide gel electrophoresis (10% gel) and transferred to Immobilon-P membrane (Millipore). Western blotting analysis was performed by using specific antibodies against PKC isoforms.

PLD Activation-- The cells were labeled with [3H]palmitic acid (3 µCi/106 cells) for 20 h, and the stimulations were carried out in the presence of 1% ethanol. At the end of the reactions, total lipids were extracted (21, 22) and phosphatidylethanol (PEt), a specific product of PLD activity, was resolved from cellular lipids by thin-layer chromatography on silica-gel G plates (Whatman), using the upper phase of a system consisting of ethyl acetate/isooctane/acetic acid/water (13:2:3:10, v/v/v/v). The lipids were identified by comparison with authentic standards run in the same plate and visualized by iodine vapors. Radioactivity was determined by liquid scintillation counting.

DAG Production-- Cells were labeled overnight with either [3H]palmitic acid (3 µCi/106 cells) or [3H]arachidonic acid (0.5 µCi/106 cells), washed, and were incubated with inhibitors for 30 min prior to stimulation with 25-50 ng/ml PMA. At the indicated times, supernatants were removed, cell monolayers were scraped, and total lipids were extracted (22). For separation of DAG, lipids were separated by thin-layer chromatography with n-hexane/diethyl ether/water (70:30:1, v/v/v). The plates were run twice in this system if monoacylglycerol determination was required as well. Radioactivity in DAG and monoacylglycerol was determined by liquid scintillation counting.

MAPK and PLA2 Immunoblotting Studies-- Cells were serum-starved for 24 h, preincubated with 100 µM propranolol for 30 min, and stimulated with 25-50 ng/ml PMA for 1 h. Cells were washed and then lysed in a buffer consisting of 1 mM Hepes, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin at 4 °C. Protein was quantified, and a 100-µg aliquot was analyzed by Western blot under conditions previously described (23), with antibodies against ERK-2 that recognizes both p42 and p44 MAPKs or against PLA2 isoforms.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phospholipase A2 Activation in Human WISH Cells-- One of the best established systems for the study of lipid mediators in amnionic cells is the human-derived cell line WISH (24). This cell line was established from altered colonies appearing in a subculture of a primary monolayer of amnion cells (18, 19). WISH cells produce large amounts of PGs after prolonged exposure to phorbol esters (18 h) (13, 20, 25). To characterize the steps in the regulation of PG production that occur during the early stages of WISH cell activation, we measured [3H]AA release in these cells after incubation with 50 ng/ml PMA for different time periods (Fig. 1A). After a time lag of approximately 30 min, significant release of [3H]AA was observed at 60 min, reaching a plateau at about 75 min. Typically, a 2-5-fold increase over basal unstimulated release was detected at an optimal PMA concentration of 25 ng/ml (Fig. 1B).


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Fig. 1.   AA release by PMA in WISH cells. [3H]AA-labeled cells were stimulated by PMA (closed circles) or vehicle (open circles). Supernatants were taken at different time points, and radioactivity was quantified by liquid scintillation counting. A, time course (25 ng/ml PMA); B, dose-response (75 min). Data are the results from three separate experiments, each carried out in triplicate. Values are shown as means ± S.E.

As a first approach, in vitro measurements of PLA2 activity in homogenates from stimulated versus unstimulated cells were conducted to identify the phospholipase A2 involved in PMA-stimulated AA release. As shown in Fig. 2A, WISH cell homogenates exhibited both Ca2+-dependent and -independent PLA2 activity. Interestingly, the Ca2+-dependent PLA2 activity was increased by a little bit less than 2-fold in homogenates from PMA-treated cells as compared with control unstimulated cell homogenates; whereas, Ca2+-independent PLA2 activity did not change (Fig. 2A). These data suggest that the PLA2 mediating PMA-induced AA mobilization is Ca2+-dependent. In keeping with these data, PMA-induced AA release by PMA was inhibited by the presence of 2 mM EGTA in the incubation medium, (Fig. 2B), demonstrating that Ca2+ is an important regulatory element in this system. We did not detect any increase in iPLA2 protein content in WISH cells or in the membrane fraction after 1 h of treatment with PMA (data not shown).


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Fig. 2.   AA release in WISH cells. A, PLA2 activity in homogenates from untreated (open bars) or PMA-treated (solid bars) WISH cells. Homogenates were prepared as described in the text, and PLA2 activity was measured in the absence or presence of calcium in the assay mix as indicated. B, effect of EGTA on AA release. [3H]AA-labeled cells were either not stimulated (Control) or stimulated with 25 ng/ml of PMA in the absence (open bars) or presence (stripped bars) of 2 mM EGTA in the incubation medium. Experiments were performed in triplicate, and the data are shown as means ± S.E.

There are two types of Ca2+-dependent PLA2 in mammalian cells, i.e. the secretory enzymes (sPLA2s) and the cytosolic Group IV PLA2 (cPLA2). An easy method for distinguishing them is to use specific inhibitors for each of these enzymes, as we have done previously in macrophages (26). Thus the effect of MAFP and LY311727, specific inhibitors of both cPLA2 and sPLA2, respectively, on PMA-stimulated AA release was analyzed. As shown in Fig. 3, MAFP strongly inhibited PMA-induced AA release, suggesting involvement of the cPLA2. On the other hand, LY311727, at concentrations up to 50 µM, was totally unable to affect the response (data not shown), ruling out a role for sPLA2 in PMA-induced AA release in WISH cells. Consistent with the latter, using reverse transcriptase-polymerase chain reaction, we have not detected significant levels of mRNA for either Group II or Group V sPLA2s in WISH cells, whether resting or treated with PMA.2 However, both the Group IV cPLA2 and the Group VI iPLA2 were easily detectable by immunoblot (see below).


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Fig. 3.   Effect of MAFP on AA release. The cells were pretreated with different concentrations of MAFP for 30 min and then stimulated with 25 ng/ml PMA (open circles) or vehicle (closed circles) for 75 min. Radioactivity in supernatants was quantified by liquid scintillation counting. Experiments were performed in triplicate and the data are shown as means ± S.E.

PAP Is Involved in PMA-induced AA Release in WISH Cells-- The effect of BEL on PMA-induced AA release was examined, and the results are shown in Fig. 4A. BEL was previously identified as a potent iPLA2 inhibitor (5), but more recent results have demonstrated its lack of specificity for iPLA2 in cells, as BEL also potently inhibits another key enzyme in lipid metabolism, i.e. the Mg2+-dependent PA phosphohydrolase (PAP) (27). In fact, the inhibitory effect of BEL on PMA-induced AA release shown in Fig. 4A cannot be attributed to iPLA2 inhibition on the basis of the results presented in Fig. 2A, which show that the iPLA2 activity does not change upon PMA treatment while the cPLA2 activity does. Moreover, BEL inhibited the PMA-induced DAG production in cells labeled with [3H]palmitic acid (Fig. 4B), indicating that BEL is indeed inhibiting the PAP. Thus, the possibility arises that the BEL effect on AA release is due to PAP inhibition. To investigate this possibility, we employed propranolol, a well established PAP inhibitor. Analogous to BEL, propranolol appreciably inhibited the PMA-induced [3H]AA release (Fig. 5A) and [3H]palmitate-labeled DAG production (Fig. 5B).


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Fig. 4.   A, effect of BEL on AA release. B, effect of BEL (5 µM) on DAG levels. The cells were pretreated with different concentrations of BEL for 30 min and then stimulated with 25 ng/ml PMA (filled bars) or vehicle (white circles) for 75 min. Free AA and DAG were quantified as described under "Materials and Methods." AA release was measured in cells prelabeled with [3H]AA. DAG was measured in cells prelabeled with [3H]palmitic acid.


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Fig. 5.   A, effect of propranolol on AA release. B, effect of propranolol (100 µM) on DAG levels. The cells were pretreated with different concentrations of propranolol for 30 min and then stimulated with 25 ng/ml PMA (closed circles) or vehicle (open circles) for 75 min. Free AA and DAG were quantified as described under "Materials and Methods." AA release was measured in cells prelabeled with [3H]AA. DAG was measured in cells prelabeled with [3H]palmitic acid.

Phospholipase D Involvement in PMA-induced AA Mobilization-- One major route for the production of the PA to be used by PAP is the PLD-mediated hydrolysis of phospholipids (28). Fig. 6A shows that, in the presence of ethanol, PMA induced the time-dependent accumulation of PEt in WISH cells, reflecting PLD activation. PEt is a specific product of PLD action in the presence of ethanol. Accumulation of PEt was detected at much earlier time points than AA release (i.e. 15 min), suggesting the possibility that products of PLD may be implicated in cPLA2 activation and attendant AA release. Should this be the case, one would expect that addition of exogenous PLD to the WISH cells would mimic the activating effect of PMA on AA release. Fig. 6, B and C, shows that treatment of the WISH cells with exogenous PLD produced a time- and dose-dependent release of [3H]AA. PLD activation by PMA was unaffected by BEL, confirming that PLD is upstream of the BEL-sensitive step, i.e. the PAP (data not shown).


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Fig. 6.   PLD activation in WISH cells. A, [3H]palmitic acid-labeled cells were stimulated with 25 ng/ml PMA (closed circles) or vehicle (open circles) in the presence of 1% ethanol for different periods of time. Lipids were extracted and separated by thin-layer chromatography, and radioactivity present in PEt was quantified by liquid scintillation counting. Data are given as percentage of radioactivity in PEt with respect to total phospholipid radioactivity. B and D, effect of purified PLD on AA release in WISH cells. [3H]AA-labeled cells were treated with different concentrations of Streptomyces chromofuscus purified PLD for 45 min (B) or with 5 units/ml PLD for different periods of time (C). Supernatants were taken, and radioactivity was quantified. Experiments were carried out in triplicate, and data are shown as means ± S.E.

A third, indirect strategy to inhibit the effect of the PAP activity is to use ethanol. By forming PEt instead of PA via phospholipase D, this alcohol depletes the substrate for PAP, thereby impairing DAG production by this route. The overall effect is thus the same as if PAP was directly inhibited. Consistent with the data with BEL and propranolol, ethanol induced a dose-dependent decrease in the PMA-induced AA release (Fig. 7). Collectively, the use of three distinct approaches to inhibit PAP activity have yielded the same result, thus underscoring the critical role that PAP plays in the chain of events leading to cPLA2 activation by PMA and, hence, to AA release in WISH cells.


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Fig. 7.   Effect of ethanol on AA release. The cells were stimulated in the presence of the indicated ethanol concentrations, and AA was quantitated as described under "Materials and Methods."

The possibility that PAP-derived DAG is serving itself as a substrate for the AA release via DAG lipase was first investigated by using the DAG lipase specific inhibitor RHC80267. This compound, at concentrations up to 100 µM, did not affect the PMA-induced AA release (not shown). However, the inhibitor slightly raised both basal and activated DAG levels in [3H]AA labeled-cells (Fig. 8A), indicating that the drug indeed prevents DAG breakdown. Examination of the time course of accumulation of DAG and MAG in [3H]AA-labeled cells did not reveal any significant variation in the levels of these two metabolites up to 60 min after PMA addition, a time point at which AA release is well underway (cf. Figs. 1A and 8, B and C).


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Fig. 8.   A, [3H]AA-containing DAG levels in control and PMA-activated cells in the absence (open bars) or presence (stripped bars) of 50 µM RHC80267. Time-course of production of [3H]AA-containing monoacylglycerol (MAG) (B) and diacylgycerol (C) in control (open circles) and PMA-activated (closed circles) cells.

Involvement of PKCalpha in PMA-induced AA Mobilization-- Activation of PKC, particularly the alpha  isoform, has previously been shown to constitute a major route for PLD activation in a wide variety of cell types (22, 28, 29). WISH cells express PKCalpha , epsilon , delta , and zeta .3 Of them, only PKCalpha was translocated to the membrane fraction after cellular activation with PMA (Fig. 9A). The translocation took place very early, being observed at 5 min and disappearing completely from the cytosolic fraction after 30 min of stimulation. To assess whether or not PKCalpha translocation to the membrane fraction was mediated by PAP-derived DAG, experiments were conducted in the presence of BEL. BEL affected neither PKCalpha binding to the membrane (Fig. 9B) nor PKC activity, as measured in vitro using a commercial kit (PKC assay system V5910, Promega) (data not shown). Like BEL, propranolol did not have any effect on PKCalpha translocation (data not shown). Involvement of PKCalpha in PMA-induced AA release was confirmed by the use of the inhibitor Gö7874, specific for Ca2+-dependent isoforms, which inhibited [3H]AA release (Fig. 9C).


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Fig. 9.   PKCalpha translocation in PMA-treated WISH cells. A, cells were stimulated with 25 ng/ml PMA for different periods of time. B, cells were pretreated with 25 µM BEL for 30 min before stimulation with PMA. Cytosolic and membrane fractions were obtained by centrifugation as described under "Materials and Methods." Protein was analyzed by Western blot using antibodies against PKCalpha . C, [3H]AA-labeled cells were preincubated with different concentrations of Gö7874 for 30 min and stimulated with 25 ng/ml PMA (closed circles) or vehicle (open circles) for 75 min. Radioactivity in the supernatants was quantified by liquid scintillation counting. Experiments were carried out in triplicate, and the data are shown as means ± S.E.

PMA-induced Phosphorylation Cascades in WISH Cells-- PMA-induced signaling events have been shown to include activation of p42/p44MAPK downstream of PKCalpha (30). In keeping with this notion, PMA was able to induce a mobility shift on SDS-polyacrylamide gel electrophoresis, indicating phosphorylation and activation of these kinases. cPLA2, which in some instances lies downstream of p42/p44MAPK (4), also experienced a mobility shift after PMA treatment (Fig. 10). Interestingly, after BEL or propranolol treatment, conditions that decrease AA release, the MAPK and cPLA2 mobility shifts were not prevented (Fig. 10). In fact, even in the absence of PMA, both inhibitors were able to induce a cPLA2 mobility shift. Moreover, neither BEL nor propranolol affected the intrinsic activity of the cPLA2 as measured in homogenates from PMA-treated cells (26; data not shown). These data indicate that inhibition of AA mobilization by PAP blockers is not due to inhibition of the signaling mechanism through which the cPLA2 increases its intrinsic specific activity, i.e. phosphorylation by MAPKs.


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Fig. 10.   Phosphorylation of p42MAPK, p44MAPK, and cPLA2. Cells were stimulated with 25 ng/ml PMA in the presence or absence of 100 µM propranolol or 25 µM BEL for 1 h. Protein was quantified and analyzed by Western blot with specific antibodies against p42/p44MAPK (ERK-2) and against cPLA2.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Very little is known about how free AA levels are regulated in the amnion, the PLA2s responsible for such a regulation, and the molecular mechanisms involved. In the present study, we have uncovered phosphatidate phosphohydrolase as a novel regulatory element within the signaling cascade that results in cPLA2 activation and AA release during the early stages of activation of the amnionic-like cell line, WISH.

BEL has recently been used as a tool to investigate whether the iPLA2 has a role in AA mobilization in different cell types, as this inhibitor possesses over 1000-fold selectivity for the iPLA2 among other PLA2 forms (31). However, BEL also inhibits the magnesium-dependent PAP (26, 32). In P388D1 cells, the IC50 for inhibition by BEL of the PAP is 8 µM, i.e. almost identical to that for inhibition of iPLA2 in the same cells (21). We have found that BEL appreciably blunts AA release in activated WISH cells; however, it blunts DAG production as well, demonstrating that the drug is affecting the PAP in addition to any effect on the iPLA2. Moreover, the inhibitory effects of BEL on AA release herein reported appear to be a consequence of PAP inhibition, since blockage of this enzyme by two other unrelated strategies, i.e. (i) direct inhibition of the enzyme by propranolol and (ii) PAP substrate depletion by ethanol, gave the same inhibitory effect on AA release. Moreover, unlike the cPLA2, the iPLA2 specific activity does not increase after cell stimulation. Our recent attempts at inhibiting iPLA2 expression by using antisense mRNA technology in WISH cells, similar to those succesfully used in P388D1 macrophages (33), have failed to detect any effect on AA release, which reinforces the notion that the iPLA2 is not an effector in this process.4 It should be noted, however, that unlike P388D1 cells, the WISH cells express very high levels of iPLA2 protein, as judged by immunoblot analysis (results not shown).

Collectively, the aforementioned results constitute, to the best of our knowledge, the first evidence implicating PAP and the metabolite it produces, DAG, in the regulation of AA mobilization. Since PAP is usually coupled to PLD, as is the case in WISH cells as well, and PLD uses phosphatidylcholine as a preferred substrate, it seems logical to assume that the DAG involved in cPLA2 activation in WISH cells arises mainly from phosphatidylcholine.

Besides the identification of PAP as an important regulator of the AA release response in WISH cells, another striking feature of the current work is the finding that none of the PAP-inhibition strategies used resulted in alteration of the intrinsic activity of the cPLA2, as measured by both phosphorylation and in vitro activity. This finding raises interesting questions as to the role of DAG in cPLA2 activation in WISH cells. The currently accepted paradigm of cPLA2 activation by stimuli considers the involvement of two different signaling branches that converge at the cPLA2 itself (4). The first one is a phosphorylation cascade that culminates in the phosphorylation of the cPLA2 and serves to increase the intrinsic activity of the enzyme. Both the nature of the kinase involved as well as the site of phosphorylation remain controversial (4). The second branch for cPLA2 signaling involves the translocation of the enzyme from the cytosol to the membrane, where its substrate is localized. This translocation, which does not modulate the cPLA2 activity itself, is currently believed to be mediated by increased Ca2+ availability although other factors may also be involved, especially in the case of stimuli like PMA which do not promote Ca2+ increases (34, 35). The two pathways for cPLA2 activation are independent of each other, but both appear to be required for proper cPLA2 activation and subsequent AA release (4).

Because PAP is not involved in regulating the cPLA2 phosphorylation cascade, it appears logical to suggest that PAP is involved in regulating binding of the cPLA2 to the membrane. DAG is long known to cause perturbations in membrane bilayers, rendering them susceptible to PLA2 attack (36). DAG accumulation in membranes has the effect of spreading apart the phospholipid headgroups, thereby making the glycerol backbone more accessible to the PLA2. Indeed, one of the most commonly utilized methods for detecting cPLA2 activity is based on this principle (20). Thus, at the same time the specific activity of the cPLA2 is increased as a result of its enhanced phosphorylation, local accumulations of DAG in the membrane allow for an appropriate substrate presentation for the enzyme. When the cPLA2 translocates to the membrane as a a result of increased Ca2+ availability and/or other factors, the enzyme will find optimal conditions to initiate AA release. The key role for DAG in this process is strengthened by the observation that, in the presence of BEL or propranolol, both p42/p44MAPK and cPLA2 become phosphorylated normally in WISH cells but no AA release is induced.

DAG is regarded as a universal activator of PKCs. However it is highly unlikely that the PAP-derived DAG is playing such a role in PMA-induced AA release in WISH cells because, in this system, the only PKC that becomes activated is the alpha  isoform, which is the one that PMA directly activates. PMA-induced PKCalpha is most likely the upstream event that triggers DAG production by activating the PLD, which in turn generates the PA substrate for the PAP. Therefore, despite the presence in WISH cells of the DAG-activable isoforms delta  and epsilon , none of them become activated after DAG levels increase. This is consistent with our suggestion that the PAP-derived DAG serves a structural, not messenger, role for AA release in PMA-activated WISH cells. In agreement with this view is the recent work by Pettitt et al. (37) in activated endothelial cells. These investigators provided compelling evidence that only the DAG which derives from inositol lipids, i.e. the one that PMA mimics in our WISH cell system, is able to activate PKC; whereas, the DAG arising from the PLD/PAP pathway does not serve a messenger role but rather a structural/metabolic role (37).

An interesting but yet unresolved question regarding AA metabolism by amnionic WISH cells relates to the fact that apreciable free AA mobilization is already detectable after a 30-min cell challenge, whereas PGE2 is released only after 4-6 h of stimulation with the phorbol ester (13). It has been suggested that COX-2 is the enzyme responsible for PG production and that its synthesis by PMA-activated WISH cells requires at least 4 h (13). Thus the question arises as to why free AA is produced much before it can be metabolized to PGE2. It seems likely that at short times, the free AA may act as a signaler rather than an intermediary metabolite. As a matter of fact, it has been suggested that exogenous AA up-regulates the expression of COX-2 in uterine stromal cells (38). The induction is not due to conversion of AA to prostaglandin by COX-1 because it occurs even in the presence of aspirin, a well known inhibitor of COX activity. Studies are currently underway in our laboratory to investigate this intriguing possibility.

In conclusion, this study has shown that PAP plays an important role in the regulation of cPLA2, possibly by facilitating interaction of the enzyme with its substrate and not by increasing the specific enzyme activity. Our data suggest the mechanism for AA mobilization in PMA-activated WISH cells depicted in Fig. 11. According to this model, the phorbol ester activates PKCalpha which, in turn, activates PLD. The PLD gives rise to PA that will be converted to DAG by PAP. The DAG produced by this pathway will act to allow a good substrate presentation for the cPLA2. Parallel to but independent of this sequence of events, PKCalpha and perhaps PA as well (39) act to activate the MAP kinase pathway, which leads to the phosphorylation of cPLA2. When these two signaling routes are turned on, the cPLA2 will start hydrolyzing phospholipids, resulting in the early generation of free AA.


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Fig. 11.   Proposed mechanism for AA release in PMA-activated WISH cells.

    FOOTNOTES

* This work was supported by Grants HD26171 and GM20501 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 619-534-3055; Fax: 619-534-7390; E-mail: edennis{at}ucsd.edu.

1 The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BEL, bromoenol lactone; cPLA2, cytosolic PLA2; DAG, diacylglycerol; iPLA2, Ca2+-independent PLA2; MAFP, methyl arachidonyl fluorophosphonate; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PAP, phosphatidate phosphohydrolase; PKC, protein kinase C; PLD, phospholipase D; PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PEt, phosphatidylethanol.

2 B. Johansen and E. A. Dennis, unpublished data.

3 M. A. Balboa and E. A. Dennis, unpublished data.

4 M. A. Balboa, J. Balsinde, and E. A. Dennis, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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