Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1*

Marita Hernández, Silvia López Burillo, Mariano Sánchez CrespoDagger , and María Luisa Nieto

From the Instituto de Biología y Genética Molecular, Universidad de Valladolid-Consejo Superior de Investigaciones Científicas, 47005 Valladolid, Spain

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such as p-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptor p-aminophenyl-alpha -D-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Phospholipases A2 (phosphatide sn-2-acylhydrolases, EC 3.1.1.4) from mammalian tissues play a role in physiological functions such as defense mechanisms and the production of bioactive lipids (1-3). In the last years, purification and molecular cloning of phospholipases A2 (PLA2)1 has allowed the characterization of several enzymes displaying significant differences in both structural and functional properties. On the one hand, the 14-kDa type IIA PLA2 (sPLA2) behaves as an acute phase protein whose production is induced in a variety of immunoinflammatory conditions, e.g. rheumatoid arthritis and endotoxemia (4-8), although its causal role in these conditions has not been ascertained, and there is no clear evidence about its involvement in the release of arachidonic acid elicited by agonists. Recent studies have shown the ability of sPLA2 to promote mitogenesis by acting on a cell surface receptor (9, 10) and the appearance of chronic epidermal hyperplasia and hyperkeratosis similar to those observed in human dermopathies in mice hyperexpressing the human type IIA PLA2 gene (11). A similar histological picture accompanied by inflammatory changes is produced by injection of sPLA2 in the skin of experimental animals (12, 13). In addition, sPLA2 may initiate cell activation because of its ability to generate the lipid mediator lysophosphatidic acid (14).

On the other hand, cytosolic phospholipase A2 (cPLA2) plays a central role in the release of arachidonic acid (AA) triggered by growth factors and neurotransmitters (15-17), and contains the consensus primary sequence (Pro-Leu-Ser-Pro) for phosphorylation by mitogen-activated protein (MAP) kinases, which play an important role in its regulation (18-20). Since sPLA2 is an ectoenzyme that first encounters the outer leaflet of the lipid bilayers, two means of interaction leading to cell signaling should be considered. (i) sPLA2 might interact with a binding structure on the outer leaflet of the cell membrane, or (ii) sPLA2 might generate both unesterified fatty acid and lysophospholipid, e.g. lysophosphatidate (LPA) and lysophosphatydylcholine, which could act on signaling either as cofactors for protein kinase C or, in the case of LPA, by acting on specific receptors. This poses as a likely possibility that sPLA2 might ultimately lead to the activation of cPLA2 by eliciting a signaling cascade mimicking the usual transducing mechanism conveyed by the physiological activators of this enzyme. In this connection, it should mentioned that cross-talk between cPLA2 and sPLA2 has been suggested in signal transduction events in polymorphonuclear leukocytes and macrophages (21, 22), and a recent study in neural cells has shown a complex interplay between neurotransmitter-activated cPLA2 and sPLA2 (23).

cPLA2 is expressed in human astrocytes of the gray matter (24), and, in a recent study, we have observed coupling of this enzyme to the activation of both muscarinic and thrombin receptors in the 1321N1 astrocytoma cell line (25, 26). This cell line displays thrombin and muscarinic M3 receptors, and its pattern of responses elicited by ligand binding includes activation of phospholipases A2, C, and D (25-32) and induction of AP-1 transcriptional activity (30, 31). 1321N1 astrocytoma cells express high amounts of cPLA2, and they do not contain sPLA2. Thus, this cell line is a good model to study the biochemical responses elicited by exogenously added sPLA2.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Plasma from patients with septicemia was obtained from venous blood anticoagulated with heparin. [9,10-3H]Myristic acid (53 Ci/mmol), [1-14C]oleate (53, 9 mCi/mmol), and [3H]arachidonic acid (100 Ci/mmol) were from Amersham International, Bucks, United Kingdom. Essentially fatty acid-free BSA was from Miles Laboratories. Reagent for the measurement of proteins according to the method of Bradford (33) was purchased from Bio-Rad. Heparin-agarose type I, p-aminophenyl-alpha -D-mannopyranoside-BSA (mannose-BSA), and porcine pancreatic PLA2 were from Sigma. A C127 mouse fibroblast line stably transfected with the coding sequence of type IIA PLA2 from human placenta (34) was used as a source of human recombinant type IIA PLA2. Rabbit polyclonal anti-cPLA2 antibody was obtained as described (35). Mouse monoclonal anti-MAP kinase antibody reacting with both p42 and p44 MAP/ERK was from Zymed Laboratories Inc., San Francisco, CA. Rabbit polyclonal anti-p38 MAP kinase antibody was from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Monoclonal anti-phosphotyrosine antibody clone 4G10 was from Upstate Biotechnology, Lake Placid, NY. The MAP kinase kinase (MEK) inhibitor PD-98059 was a gift from Dr. Alan R. Saltiel (Parke Davis Pharmaceutical Research, Ann Arbor, MI) (36). The p38 MAP kinase inhibitor SB 203580 was a gift from Dr. John C. Lee (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) (37). Glutathione S-transferase (GST) fusion protein with amino acids 1-223 of the N-terminal portion of c-Jun protein (a kind gift of Dr. Carmen Caelles, Instituto de Investigaciones Biomédicas, Madrid, Spain) was expressed in bacteria using a pGEX-2T plasmid (Pharmacia Biotech Inc.) and purified with glutathione-agarose beads from Sigma.

Purification of sPLA2-- sPLA2 was purified from both plasma of patients with septicemia and culture medium according to the protocol described in Ref. 38. Briefly, heparin-agarose was used to bind sPLA2 from plasma. Fractions showing PLA2 activity in the [1-14C]oleate-labeled Escherichia coli assay were concentrated and loaded into a HiLoad Superdex 75 column (Pharmacia LKB, Uppsala, Sweden). Fractions containing PLA2 after this step were made in 0.1% trifluoroacetic acid, and applied into a C1/C8 reverse-phase FPLC column (ProRPC HR 5/2, Pharmacia LKB). Fractions showing PLA2 activity were pooled and evaporated to dryness in a Speed-Vac concentrator. Human recombinant type IIA phospholipase A2 was purified from cultures at superconfluence of line C127 mouse fibroblasts stably transfected with the coding sequence of type IIA PLA2 from human placenta (34).

Assay of sPLA2 Activity-- The assay was carried out in a total volume of 0.1 ml, according to the procedure of Elsbach et al. (39). Samples were incubated with approx 5,000 dpm of [1-14C]oleate-labeled autoclaved E. coli of a K12 strain, containing 10-20 nmol of phospholipid, as assessed by the measurement of phospholipid-associated phosphate. The assay medium contained 0.1 M Tris/HCl, 1 mg/ml fatty acid-free BSA, and 0.5 mM CaCl2, pH 7.4. The reaction proceeded for 30 min and was stopped by addition of 0.04 ml of ice-cold 2 N HCl and 0.02 ml of 10% BSA, followed by centrifugation for 5 min at 13,000 rpm in an Eppendorf microcentrifuge. The radioactivity released into the supernatant was assayed by liquid scintillation counting.

Cell Culture and Metabolic Labeling of 1321N1 Cells-- 1321N1 astrocytoma cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal calf serum at 37 °C in an atmosphere containing 5% CO2. Labeling with [3H]AA was performed in cells in monolayer that had been deprived of fetal calf serum for 16 h to render them quiescent. Labeling with [3H]AA was carried out for 2 h in the presence of 0.3 µCi of [3H]AA/ml. Labeling with 0.3 µCi/ml [1-14C]oleate and 1 µCi/ml [3H]myristic acid was carried out under similar conditions but increasing the labeling period to overnight incubation. After labeling, cells were washed at 37 °C four or five times with medium, and finally allowed to equilibrate at 37 °C before addition of agonists or vehicle solution. The release of labeled [3H]AA and [1-14C]oleic acid acid was assessed in 0.2-ml aliquots of culture medium. Production of LPA was assessed from the incorporation of [3H]myristic acid into phosphatidic acid and was separated from the label incorporated in other phospholipid classes by two-dimensional chromatography using a system of solvents consisting of chloroform/methanol/28% ammonium hydroxide (6:4:1; v/v/v) in the first dimension and chloroform/acetone/methanol/acetic acid/water (6:8:2:2:1; v/v/v) in the second dimension (40). Experiments were carried out with triplicate samples.

Measurement of DNA Synthesis Reinitiation-- Quiescent 1321N1 cells were treated in serum-free Dulbecco's modified Eagle's medium for 24 h with different agonists in the presence of 0.5 µCi/ml [3H]thymidine. At the end of this period, the incubation was terminated with three washes with ice-cold 0.1 M MgCl2, and the radioactivity incorporated into the trichloroacetic acid-precipitable fraction measured.

Immunoblots of cPLA2, p42 MAP Kinase, and Immunoprecipitated p38 MAP Kinase-- Cell lysates from preconfluent 1321N1 cells were loaded into a 10% SDS-PAGE gel, and transferred to polyvinyldifluoride membrane (Immobilon P, Millipore Corp., Bedford, MA) using a liquid transfer module from CBS Laboratories. The membranes were blocked with dry milk for 2 h, washed with Tris-buffered saline, and used for immunoblot using a rabbit polyclonal anti-cPLA2. When the purpose of the experiments was the detection of p42 MAP kinase, a semidry transfer system was used and the membrane was incubated with mouse monoclonal antibody. This was followed by incubation with sheep anti-mouse IgG-horseradish peroxidase-conjugated antibody, and detection with the Amersham ECL system. For detection of tyrosine phosphorylation of p38 MAP kinase, the endogenous kinase was immunoprecipitated from cell lysates using anti-p38 MAP antibody. The immune complex was recovered using Gammabind G-Sepharose. After washing three times with Nonidet-P-40-buffer and twice with LiCl buffer, the beads were resuspended in Laemmli sample buffer and subjected to SDS-PAGE. The extent of tyrosine phosphorylation of the p38 MAP kinase immunoprecipitated was determined by immunoblot with anti-phosphotyrosine mouse monoclonal antibody.

Assay of JNK Activity-- To obtain the substrate for the kinase assay as a GST-c-Jun fusion protein, the procedure of Smith and Corcoran (41) was followed. For this purpose, transformed XL1-blue cells containing a pGEX-2T plasmid encoding residues 1-223 of the N-terminal portion of c-Jun protein were grown in LB/ampicillin medium. The expression of the fusion protein was induced by addition of 1 mM isopropyl-1-thio-beta -D-galactoside. Cells were lysed using a probe sonicator and the fusion protein purified with glutathione-agarose beads. The cytosolic extracts for the kinase assay were obtained from the lysis of 5 × 106 1321N1 cells in 200 µl of a medium containing 25 mM Hepes, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 100 mM orthovanadate, 20 mM beta -glycerophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, pH 7.7. After centrifugation at 12,000 rpm at 4 °C, the supernatant was diluted in 600 µl of the above mentioned medium without NaCl, and mixed with 10 µg of GST-c-Jun protein and glutathione-agarose beads. The mixture was incubated under continuous shaking for 3-5 h at 4 °C, and then washed to remove the fraction not associated to the glutathione-agarose beads. The kinase reaction was carried out with 20 mM ATP and 5 µCi of [gamma -32P]ATP in a volume of 30 µl. The reaction was diluted in buffer and centrifugated to discard supernantant and then boiled in Laemmli SDS sample buffer and DTT. Phosphorylated GST-c-Jun was resolved by 10% SDS-PAGE and detected by autoradiography. Quantitation of the phosphorylation was carried out by densitometric scanning.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

sPLA2 Produces [3H]AA Release and Mitogenesis in 1321N1 Astrocytoma Cells, but Does Not Release [1-14C]Oleic Acid-- Incubation of 1321N1 cells with sPLA2 at concentrations of 10 ng to 0.4 µg induced the release of [3H]AA into the medium (Table I, Fig. 1A). This release was similar to that produced by agonists acting on membrane receptors on this cell line, namely carbachol (25), thrombin (26), and LPA (Fig. 1B). Astrocytes labeled with [1-14C]oleic acid were treated with sPLA2 under the same conditions used for the assay of [3H]AA release. As shown in Table I, no significant release of [1-14C]oleic acid was observed. Since sPLA2 produces mitogenesis in astrocytes (9), we addressed whether this response was also elicited in quiescent 1321N1 cells, using 10% fetal calf serum as a positive control and thrombin as a prototypic mitogenic agonist of this cell line (42). As shown in Table II, sPLA2 behaved as a mitogenic agonist somewhat more potent than thrombin.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of sPLA2 on [3H]AA and [1-14C]oleate release by 1321N1 cells
1321N1 cells were labeled and incubated for 45 min at 37 °C with different concentrations of sPLA2. At the end of this period, the radioactivity contained in 0.2 ml of cell medium was assayed. The radioactivity incorporated in cell phospholipids under the conditions used for labeling was approx 120,000 dpm for [3H]AA and approx 16,000 for [1-14C]oleate. Results represent mean ± S.E. of three independent experiments in triplicate.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Release of [3H]AA in response to sPLA2 and LPA. 1321N1 cells were incubated in the presence of 0.1 µg/ml sPLA2 (A) or 0.2 µM LPA (B) for the times indicated, and then the release of [3H]AA assayed in the culture medium. Closed circles indicate cells incubated in the presence of agonist. Open circles indicate cells incubated in the presence of vehicle. Data represent mean ± S.E. of six independent experiments in duplicate. *, p < 0.05 as compared with control cells incubated for the same time. The closed triangle in A indicates cells preincubated for 30 min with 25 µM PD-98959 prior to the addition of sPLA2. The closed square in A indicates cells pretreated with 25 µM SB 203580 under identical conditions. The release of [3H]AA in the presence of these drugs has been compared with that produced by sPLA2 in the absence of these compounds.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Incorporation of [3H]thymidine into acid-precipitable material
Results are expressed as percent of the response elicited by 10% FCS (83,000 dpm). Data represent mean ± S.E. of three experiments with duplicate samples.

sPLA2 Induces the Phosphorylation of Both cPLA2 and MAP Kinases-- Since cPLA2 is the most specific enzyme that releases arachidonate from phospholipids, and sPLA2 does not display selectivity for any unsaturated fatty acid on the sn-2 position of phospholipids (43, 44), sPLA2 responses might a priori reflect either a direct consequence of its catalytic activity or recruitment of the arachidonate-selective enzyme cPLA2. Considering that 1321N1 cells contain cPLA2 as the main PLA2 activity detected in cell-free homogenates and the implication of this activity in the mobilization of [3H]AA produced by receptor stimulation (25, 26), we hypothesized that activation cPLA2 could explain the release of [3H]AA triggered by sPLA2. The increase in catalytic activity of the 85-kDa PLA2 has been linked to phosphorylation of the enzyme, which results in reduced mobility upon electrophoresis. As shown in Fig. 2A, 0.1 µg/ml sPLA2 purified from the plasma of patients with septicemia induced phosphorylation of cPLA2. The activation of this protein shows a time course that clearly precedes [3H]AA release. Maximal amount of P-cPLA2 was achieved within 10-15 min and was maintained up to 30 min after cellular stimulation. Interestingly, phosphorylation of the p42 MAP kinase preceded cPLA2 phosphorylation, since it was near maximal values at 5 min (Fig. 2A). In vitro kinase assay of c-Jun kinase activity in lysates from cells stimulated with sPLA2 showed an increase of the activity that peaked about 10 min after addition of sPLA2 (Fig. 3A). Blotting with anti-phosphotyrosine antibody of the immunoprecipitate obtained with anti-p38 MAP antibody in lysates from 1321N1 cells, showed an increase of tyrosine phosphorylation of p38 MAP kinase of about 4-fold, 2 min after addition of sPLA2 (Fig. 3B), thus suggesting that sPLA2 activates all the subgroups of the MAP kinase family following different time courses.


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 2.   Activation of cPLA2 and p42 MAP kinase by sPLA2 (A) and LPA (B). Cells were stimulated with 0.1 µg/ml sPLA2 purified from patients with septicemia or 0.2 µM LPA, and at the times indicated washed and then lysed in Laemmli's buffer. About 50 µg of protein from the same samples were processed separately by SDS-PAGE (10% gel) with prolonged electrophoresis to separate the phosphorylated (P-cPLA2, P-p42-MAP) from the nonphosphorylated forms (cPLA2, p42-MAP) of cPLA2 and p42 MAP. The proteins were electrophoretically transferred to polivinyldifluoride membranes, and immunoblotting was carried out with anti-cPLA2 antibody or with anti-MAP/ERK antibody.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of sPLA2 on c-Jun kinase activity 2nd on p38 MAP kinase phosphorylation. 1321N1 cells were incubated with sPLA2 for the times indicated, and at the end of these periods, cell lysates were collected and used for in vitro assay of c-Jun kinase activity using GST-c-Jun protein and glutathione-agarose beads. Heat shock (HS) was used as a positive control for c-Jun kinase activation. This was carried out by incubating the cells for 2 min in medium preheated at 45 °C, followed by incubation at 37 °C. Quantitative analysis of the phosphorylation of GST-c-Jun was obtained by densitometric scanning and is expressed as fold-increase of basal activity (A). The experiment shown in B was carried out by immunoprecipitation of p38 MAP kinase from cell lysates, SDS-PAGE separation of the immunoprecipitate, and blotting with anti-phosphotyrosine antibody. The histogram shows the densitometric scanning of the blots. P-Y-p38-MAP indicates p38 MAP kinase phosphorylated in tyrosine.

We also investigated the effect of human recombinant sPLA2 isolated from permanent transfected C127 fibroblasts. Stimulation of astrocytes with concentrations of human recombinant sPLA2 above 0.1 µg/ml also resulted in a shift of the electrophoretic mobility of cPLA2 (Fig. 4). Similarly, the addition of type I PLA2 (pancreatic PLA2, 0.8-8 µg/ml) to 1321N1 astrocytoma cells also increased the amount of P-cPLA2 detected upon electrophoresis (Fig. 4). To confirm that the observed increase of the cPLA2 phosphorylation was due to sPLA2 rather than linked to a possible lipopolysaccharide contamination in the sPLA2 preparation from septic patients, we treated our cells with 10 µg/ml lipopolysaccharide. SDS-PAGE revealed that lipopolysaccharide is not able to induce cPLA2 phosphorylation (data not shown), thus ruling out the view that the observed activation of cPLA2 could be due to contamination by lipopolysaccharide. Having established that addition of either of the two secreted forms of PLA2 induced phosphorylation of cPLA2, we hypothesized two possible mechanisms either a direct action of sPLA2 on its receptor or an indirect effect through lipid mediators generated as a consequence of its catalytic activity.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4.   Activation of cPLA2 by pancreatic PLA2, recombinant human sPLA2, and LPA: dose dependence. 1321N1 cells were incubated with different concentrations of agonists for 15 min. At the end of these periods, the cell lysate was processed. Immunoblots are representative of at least three blots with identical pattern.

sPLA2 Elicits Its Effect Independently of Lysophosphatidate Formation-- Since LPA is a mitogenic agonist (reviewed in Ref. 45) and a product of sPLA2 (14), we put forward the hypothesis that sPLA2 could elicit its effect via the formation of this lipid mediator that acts via the interaction with a G-protein-coupled receptor. To check this notion, we first looked at the effect of LPA. As shown in Fig. 1B, a concentration of LPA as low as 0.2 µM induced [3H]AA release. To determine the time course of LPA-induced phosphorylation of cPLA2, astrocytes were exposed to 0.2 µM LPA for 0-60 min. As shown in Fig. 2B, the response is already evident by 5 min and is fully developed by 10 min. cPLA2 band-shift induced by LPA was preceded by p42 MAP kinase phosphorylation, which was already significant at 1 min and maximal at 5 min (Fig. 2B). Fig. 4 shows the dose-dependent effect of LPA. Since both sPLA2 and LPA produced a similar pattern of activation, this finding could be considered as an initial hint that LPA could be involved in the mediation of sPLA2 effect.

It has been shown that the LPA-induced MAP kinase activation is sensitive to pertussis toxin inhibition (46, 47), thus indicating a critical role for a pertussis toxin-sensitive Gi-protein. On this basis, if sPLA2 were acting through LPA generation, cPLA2 and MAP kinase activation in response to sPLA2 should show identical sensitivity to PTX. Then, in astrocytes preincubated with or without 100 nM PTX, we looked at the effect of sPLA2 on the phosphorylation of p42 MAP kinase and cPLA2. Whereas overnight incubation of 1321N1 cells with PTX inhibited the LPA-induced shift in electrophoretic mobility of both cPLA2 and p42 MAP kinase, this treatment did not affect the ability of sPLA2 to phosphorylate cPLA2 or p42 MAP kinase (Fig. 5). This suggests not only that LPA is not involved in the cellular response to sPLA2, but also that sPLA2 acts through a pathway independent of Gi-proteins.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of PTX on the activation of cPLA2 and p42 MAP kinase elicited by LPA and sPLA2. 1321N1 cells were incubated overnight with 100 ng/ml PTX, and then stimulated with vehicle or the indicated concentrations of agonists for 15 min. At the end of this period, the cell lysate was obtained and used to separate, by electrophoresis, the phosphorylated and nonphosphorylated forms of cPLA2 and p42 MAP. Immunoblot is representative of two blots with identical trend.

Attempts to demonstrate formation of LPA by sPLA2 were carried out by labeling the phospholipid pools with [3H]myristic acid and analysis of the cellular culture medium. The lipid fraction was analyzed by a two-dimensional TLC system, which allows LPA to be separated from other polar lipids with a high degree of resolution. However, upon sPLA2 treatment, [3H]LPA accumulation was not detected, even though a high concentration of lipid-free BSA was added to the medium to trap LPA because of its strong binding to albumin (48).

Inactivation of sPLA2 Catalytic Activity Does Not Block the Ability to Induce Phosphorylation of cPLA2-- As we failed to find accumulation of LPA or any other fatty acid but [3H]AA in the cell culture medium, we addressed the possibility of regarding sPLA2 as the direct responsible for cPLA2 phosphorylation. To determine whether blockade of sPLA2 catalytic activity may affect its ability to induce cPLA2 activation, the actions of known sPLA2 inhibitors were examined. We first looked at the effect of p-bromophenacyl bromide (BPB), a compound that inactivates the enzyme by alkylating a histidine residue located in the active site (49). Pretreatment of sPLA2 for 30 min with different doses of the inhibitor resulted in a dose-dependent lose of its catalytic activity on the E. coli membrane system, reaching a complete blockage at 100 µM. However, even in the presence of this doses of BPB, the cPLA2 band-shift induced by sPLA2 was not affected (Fig. 6). It should be noted that 1 mM BPB (but not the other doses) alters agonist-induced cPLA2 band-shift, thus suggesting a toxic effect of this compound, which we could confirm by the appearance of the cell culture; this agrees with the report by Lister et al. (50), who have suggested that the inhibitory effects of high concentrations of BPB is nonspecific, as it is due to the hydrophobicity of the compound. Incubation of sPLA2 for 30 min with the thiol reagent DTT, dramatically reduced the catalytic activity of this enzyme (50% with 0.1 mM, 90% with 1 mM, and 100% with 10 mM); however, this treatment did not affect the ability of sPLA2 to phosphorylate cPLA2 upon addition to 1321N1 cells (Fig. 6). Taken together, the above results show that both cPLA2 phosphorylation and AA mobilization induced by sPLA2 are events independent of the catalytic activity of the enzyme.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of BPB, DTT, and heparin on the activation of cPLA2 by sPLA2. BPB was added to the cells in the range of concentrations shown, 10 min before the addition of sPLA2. In the case of experiments with DTT, the enzyme was incubated with the indicated concentrations of DTT for 30 min at room temperature, and then added to the cells to reach a final 100-fold dilution of DTT, and the indicated concentration of sPLA2. The left lane shows the effect of the maximal concentration of DTT on 1321N1 cells. Heparin was added to 1321N1 cells 10 min before sPLA2.

Compounds Blocking Binding of sPLA2 to Cell Membrane Surface Inhibit the Ability to Phosphorylate cPLA2-- In contrast to the aforementioned data, previous treatment of 1321N1 cells with mannose-BSA prior to sPLA2 addition blocked cPLA2 band-shift (Fig. 7) with an EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor (51). The same effect was observed when the same samples were used to study the effect on p42 MAP kinase band-shift (Fig. 7). Since it has been described that sPLA2 may trigger mast cell activation through binding of its heparin-binding domain to the cell surface (52), the effect of exogenous heparin on sPLA2-induced cPLA2 phosphorylation was also tested. As shown in the lower panel in Fig. 6, concentrations of heparin similar to those active on mast cells inhibited the cPLA2 band-shift, without affecting significantly sPLA2 catalytic activity on [1-14C]oleate-labeled autoclaved E. coli (data not shown). All these findings suggesting that both cPLA2 and p42 MAP kinase activation can be explained by interaction of sPLA2 with a binding structure on 1321N1 cell surface.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of mannose-BSA on the activation of cPLA2 and p42 MAP kinase elicited by sPLA2. 1321N1 cells were incubated for 10 min with the indicated concentrations of mannose-BSA prior to the addition of sPLA2. This immunoblot is representative of three blots with identical pattern.

Blockade of MAP/ERK Kinase Activation Inhibits cPLA2 Phosphorylation and [3H]AA Release-- As shown previously, prolonged SDS-PAGE and immunoblotting of 1321N1 cell lysates, with a monoclonal antibody that recognizes an epitope found in both the 42- and 44-kDa isoforms of MAP/ERK kinases, only showed positive staining of a 42-kDa protein in resting cells, suggesting that this is the main isoform of the ERK subfamily of MAP kinases expressed in 1321N1 cells. Preincubation of 1321N1 cells with the compound PD-98059 (36), which inhibits MAP/ERK kinase activation by interfering with the upstream kinase MEK, inhibited both cPLA2 and p42 MAP kinase activation over the same range of concentrations (Fig. 8), as well as the release of [3H]AA (Fig. 1A), thus suggesting the involvement of the MAP/ERK subgroup of MAP kinases in the phosphorylation of cPLA2 elicited by sPLA2. Pretreatment of the cells with the p38 inhibitor SB 203580 at the concentration of 25 µM also caused inhibition of [3H]AA release (Fig. 1A), thus suggesting that this subfamily of MAP kinases could be involved in the pathway for cPLA2 activation elicited by sPLA2.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of the MEK inhibitor PD-98059 on the activation of cPLA2 and p42 MAP kinase elicited by sPLA2. 1321N1 cells were incubated for 10 min with different concentrations of PD-98059 prior to the addition of sPLA2. About 10 min after addition of sPLA2, the cell lysate was collected for immunoblotting. This immunoblot is representative of two blots with identical trend.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have selected 1321N1 astrocytoma cells to study the effect of sPLA2 because these cells do not express this enzyme, but do contain high amounts of cPLA2, which is the form of enzyme most usually involved in the release of [3H]AA coupled to receptor stimulation. Analysis of the physiological effects of sPLA2 indicates several possible mechanisms through which they might be exerted. One of them takes into account the lysophospholipids formed as a consequence of the catalytic properties of the enzyme. In this connection, analysis of the involvement of LPA is of central importance, since this is a multifunctional signaling phospholipid that elicits cell responses by binding to a receptor, which couples to both PTX-sensitive and PTX-insensitive G-proteins (Gi and Gq, respectively) to trigger various effector pathways. At least four G-protein-mediated signaling pathways have been identified in the action of LPA (revised in Ref. 45): (i) stimulation of phospholipases C, D, and A2 (this report); (ii) inhibition of adenylyl cyclase; (iii) activation of Ras and the downstream Raf/MAP kinase pathway; and (iv) protein-tyrosine phosphorylation. This is relevant to the present study since LPA is detected in human serum at concentrations in the range 2-70 µM (45-47), and the effect of sPLA2 on platelets incubated with lipid microvesicles has been related to the production of LPA (14). Some of our findings agree with this mechanism of signaling in view of the ability of exogenous LPA to trigger biochemical signals in 1321N1 cells resembling a portion of the effect of sPLA2; however, a careful appraisal of the results shows the existence of several differences, e.g. the involvement of a PTX-sensitive G-protein in LPA signaling, which is not involved in sPLA2 signaling. Moreover, direct attempts to assay LPA formation upon sPLA2 did not show the production of this mediator. Generation of unesterified fatty acids by sPLA2 could be another mechanism through which this enzyme conveys cell responses. This point is a matter of considerable debate, since there is a number of mammalian cells where there has not been possible to trigger AA release by sPLA2 (38, 53, 54), unless a membrane rearrangement of phospholipids is produced (55). Separation by TLC of cell-associated lipids and assay of supernatants of cells in culture stimulated with sPLA2 showed no evidence of unesterified [1-14C]oleate, but did show [3H]AA. Since unlike cPLA2, sPLA2 does not have a preferential effect on AA-containing membrane phospholipids as compared with those containing other fatty acids, our results should be explained on the basis of the activation by sPLA2 of a selective mechanism for AA release that would implicate a signaling cascade leading to cPLA2 activation. Selective release of AA by sPLA2 has already been reported in mice bone marrow mast cells (56). In this study, concentrations of approx 1 µg/ml PLA2 from different sources, including human recombinant type IIA sPLA2 and Naja naja type I PLA2, elicited the release of AA in a similar way to that observed in response to immunological challenge by specific antigen. Since other unsaturated fatty acids were not detected in the supernatant, this finding also points to the recruitment by sPLA2 of a selective mechanism of AA release.

The characterization of the binding site in cell membrane involved in the triggering of the response to sPLA2 herein described requires a detailed discussion in view of the different structures that could be involved. Thus, there is some evidence associating many effects of sPLA2 to the activation of a membrane surface receptor, which shows significant homology with the macrophage mannose-binding receptor (6, 7, 51), and is also activated by the pancreatic type of PLA2, thus suggesting that endogenous PLA2(s) might be its physiological ligands. In fact, stimulation of prostaglandin synthesis by pancreatic type PLA2 acting through a receptor-binding reaction has been shown in rat mesangial cells (57) and in mouse osteoblastic cells (58). Moreover, inflammatory factors stimulate expression of type IIA PLA2 in astrocytes in culture (59), and brain tissue is rich in N-type PLA2 receptors (60). However, previous reports do not support the involvement of PLA2 receptors in our system, since unlike the rabbit receptor (10), the human 180-kDa receptor expressed in COS cells binds neither type IIA PLA2 nor mannose-BSA (61). Interaction of sPLA2 with heparan sulfate proteoglycans is another possibility, in view of a recent report where sPLA2 expressed endogenously and anchored on cell surfaces via its C-terminal heparin-binding domain was shown to be involved in the biosynthesis of prostaglandins elicited by growth factors and cytokines (62). Our attempts to unveil the binding structure by using pharmacological agents such as heparin and mannose-BSA have shown inhibition by either compound, thus suggesting more than one binding structure or, alternatively, a scarce selectivity for these compounds. Therefore, additional studies of binding and receptor expression are required to characterize these structures more precisely.

Irrespective of the nature of the membrane structure involved in sPLA2 binding, the overall response induced by sPLA2 in 1321N1 cells is in keeping with a mechanism dependent on the occupancy of the physiological binding sites for secreted PLA2. Little is known about the biochemical signaling triggered by sPLA2 receptor binding. Murakami et al. (52) have proposed the involvement of protein-tyrosine phosphorylation reactions in sPLA2-mediated mast cell activation in view of the blockade of the response by inhibitors of this reactions such as genistein and herbimycin A. In view of the important interactions of protein-tyrosine phosphorylation signaling and activation of the MAP kinase cascade, this report agrees with our finding of the activation of the MAP kinase cascades by sPLA2, including p42 MAP kinase, p38, and c-Jun kinase. Based on the different time courses of the activation of these kinases, and the results obtained with the MEK inhibitor PD-98959 as well as with SB 203580, which inhibits p38 MAP kinase activity, our data suggest the implication of both kinases in the signaling pathway leading to [3H]AA release, although characterization of the actual kinase(s) implicated in cPLA2 phosphorylation requires additional studies. Studies on the regulation of cPLA2 have stressed the requirement of Ca2+-dependent translocation to the cell membrane for elicitation of its catalytic effect (63). In keeping with this mechanism, we have observed that both pancreatic type PLA2 and type IIA sPLA2 elicit Ca2+ mobilization in fura-2-labeled 1321N1 cells2 showing a pattern similar to that elicited by LPA, thus suggesting a mechanism of action compatible with the occupancy of a binding site. However, since mechanisms other than Ca2+ mobilization have been implicated in the translocation of cPLA2 (25, 64), we cannot establish a direct link between Ca2+ mobilization and the activation of cPLA2 as yet.

As to the pathophysiological significance of our findings, it should be pointed out that the effect of sPLA2 has been obtained with concentrations of enzyme below those detected in human plasma in a number of clinical conditions, including septic shock (65), salicylate intoxication (66), and severe Plasmodium falciparum malaria (67).

In summary, our data show that sPLA2 elicits biochemical signaling in 1321N1 astrocytoma cells by a mechanism that is best explained by interaction with a membrane receptor similar to the macrophage mannose receptor or, alternatively, via engagement of heparan sulfate proteoglycans. The set of responses observed includes phosphorylation of cPLA2, most probably involving the p42 MAP kinase route, release of AA, and mitogenesis. These findings might be of interest to explain some of the controversial findings regarding release of AA by sPLA2.

    ACKNOWLEDGEMENTS

We thank Dr. G. Bereziat for providing the transfected fibroblast cell line, Dr. Victor Calvo for help in assaying MAP kinase, Dr. J. Castañeda for selecting patients with multiple organ failure and high levels of sPLA2 to obtain plasma, Dr. Joan Heller Brown for providing the 1321N1 cell line, and Dr. Carmen Caelles for providing the GST-c-Jun fusion protein.

    FOOTNOTES

* This study has been supported by Grant PB94-1114 from Comisión Interministerial de Ciencia y Tecnología.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: Instituto de Biología y Genética Molecular, Facultad de Medicina, 47005 Valladolid, Spain. Tel.: 34-83-423273; Fax: 34-83-423085; E-mail: mscres{at}wamba.cpd.uva.es.

1 The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BPB, p-bromophenacyl bromide; BSA, bovine serum albumin; cPLA2, cytosolic phospholipase A2; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; LPA, lysophosphatidic acid; MAP, mitogen-activated protein; mannose-BSA, p-aminophenyl-alpha -D-mannopyranoside-BSA; MEK, mitogen-activated protein kinase kinase; PAGE, polyacrylamide gel electrophoresis; PTX, pertussis toxin; sPLA2, secretory phospholipase A2.

2 M. Hernández, S. López Burillo, M. Sánchez Crespo, and M. L. Nieto, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. (1991) FASEB J. 5, 2068-2077[Abstract/Free Full Text]
  2. Mayer, R. J., and Marshall, L. A. (1993) FASEB J. 7, 339-348[Abstract/Free Full Text]
  3. Serhan, C. N., Haeggstrom, J. Z., and Leslie, C. C. (1996) FASEB J. 10, 1147-1158[Abstract/Free Full Text]
  4. Vadas, P., Wasi, S., Movat, H., and Hay, J. H. (1981) Nature 293, 583-585[Medline] [Order article via Infotrieve]
  5. Vadas, P. (1984) J. Lab. Clin. Med. 104, 873-881[Medline] [Order article via Infotrieve]
  6. Chilton, F. (1996) J. Clin. Invest. 97, 2161-2162[Free Full Text]
  7. Hack, C. E., Wolbink, G. J., Schalwijk, C., Speijer, H., Hermens, W. T., van den Bosch, H. (1997) Immunol. Today 18, 111-115[CrossRef][Medline] [Order article via Infotrieve]
  8. Jacques, C., Béréziat, G., Humbert, L., Olivier, J. L., Corvol, M. T., Masliah, J., Berenbaum, F. (1997) J. Clin. Invest. 99, 1864-1872[Abstract/Free Full Text]
  9. Arita, H., Hanasaki, K., Hanako, T., Oda, S., Teraoka, H., and Matsumoto, K. (1991) J. Biol. Chem. 266, 19139-19141[Abstract/Free Full Text]
  10. Lambeau, G., Ancian, P., Barhanin, J., and Lazdunski, M. (1994) J. Biol. Chem. 269, 1575-1578[Abstract/Free Full Text]
  11. Grass, D. S., Felkner, R. H., Chiang, M. Y., Wallace, R. E., Nevalainen, T. J., Bennet, C. F., Swanson, M. E. (1996) J. Clin. Invest. 97, 2233-2241[Abstract/Free Full Text]
  12. Pruzanski, W. P., Vadas, P., and Fornasier, V. (1986) J. Invest. Dermatol. 86, 380-382[Abstract]
  13. Nair, X., Nettleton, D., Clever, D., Tramposch, K. M., Ghosh, S., Franson, R. C. (1993) Inflammation 17, 205-215[Medline] [Order article via Infotrieve]
  14. Fourcade, O., Simon, M. F., Viode, C., Rugani, N., Leballe, F., Ragab, A., Fournie, B., Sarda, L., and Chap, H. (1995) Cell 80, 919-927[Medline] [Order article via Infotrieve]
  15. Leslie, C. C., Voelker, D. R., Channon, J. Y., Wall, M. M., Zelarney, P. T. (1988) Biochim. Biophys. Acta 963, 476-492[Medline] [Order article via Infotrieve]
  16. Clark, J. D., Lin, L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., Knopf, J. L. (1991) Cell 65, 1043-1051[Medline] [Order article via Infotrieve]
  17. Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., Kramer, R. (1991) J. Biol. Chem. 266, 14850-15853[Abstract/Free Full Text]
  18. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556[Free Full Text]
  19. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846[Free Full Text]
  20. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., Davis, R. J. (1993) Cell 72, 269-278[Medline] [Order article via Infotrieve]
  21. Wijkander, J., O'Flaherty, J. T., Nixon, A. B., Wykle, R. L. (1995) J. Biol. Chem. 270, 26543-26549[Abstract/Free Full Text]
  22. Balsinde, J., and Dennis, E. A. (1996) J. Biol. Chem. 271, 6758-6765[Abstract/Free Full Text]
  23. Kolko, M., DeCoster, M. A., Rodríguez de Turco, E. B., Bazán, N. G. (1996) J. Biol. Chem. 271, 32722-32728[Abstract/Free Full Text]
  24. Stephenson, D. T., Manetta, J. V., White, D. L., Chiou, X. G., Cox, L., Gitter, B., May, P. C., Sharp, J. D., Kramer, R. M., Clemens, J. A. (1994) Brain Res. 637, 97-115[CrossRef][Medline] [Order article via Infotrieve]
  25. Bayón, Y., Hernández, M., Alonso, A., Núñez, L., García-Sancho, J., Leslie, C. C., Sánchez Crespo, M., Nieto, M. L. (1997) Biochem. J. 323, 281-287[Medline] [Order article via Infotrieve]
  26. Hernández, M., Bayón, Y., Sánchez Crespo, M., Nieto, M. L. (1997) Biochem. J. 328, 263-269[Medline] [Order article via Infotrieve]
  27. Orellana, S. A., Solski, P. A., and Heller Brown, J. (1985) J. Biol. Chem. 260, 5236-5239[Abstract]
  28. Martinson, E. A., Goldstein, D., and Brown, J. H. (1989) J. Biol. Chem. 264, 14748-14754[Abstract/Free Full Text]
  29. Trejo, J. A., and Brown, J. H. (1991) J. Biol. Chem. 266, 7876-7882[Abstract/Free Full Text]
  30. LaMorte, V. J., Kennedy, E. D., Collins, L. R., Goldstein, D., Harootunian, A. T., Brown, J. H., Feramisco, J. R. (1993) J. Biol. Chem. 268, 19411-19415[Abstract/Free Full Text]
  31. Martinson, E. A., Trilivas, I., and Brown, J. H. (1990) J. Biol. Chem. 265, 22282-22287[Abstract/Free Full Text]
  32. Nieto, M. L., Kennedy, E., Goldstein, D., and Heller Brown, J. (1994) Mol. Pharmacol. 46, 406-413[Abstract]
  33. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  34. Pernas, P., Masliah, J., Olivier, J. L., Salvat, C., Bereziat, G. (1991) Biochem. Biophys. Res. Commun. 178, 1298-1305[Medline] [Order article via Infotrieve]
  35. deCarvalho, M. S., McCormack, F. X., Leslie, C. C. (1993) Arch. Biochem. Biophys. 306, 534-540[CrossRef][Medline] [Order article via Infotrieve]
  36. Dudley, D. T., Pang, L., Decker, S.. J., Bridges, A. J., Saltiel, A. R.. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract]
  37. Lee, J. C., and Young, P. R. (1996) J. Leukocyte Biol. 59, 152-157[Abstract]
  38. Gijón, M. A., Pérez, C., Méndez, E., and Sánchez Crespo, M. (1995) Biochem. J. 306, 167-175[Medline] [Order article via Infotrieve]
  39. Elsbach, P., Weiss, J., Franson, R. C., Beckerdite-Quagliata, S., Schneider, A., Harris, L. (1979) J. Biol. Chem. 254, 11000-11009[Abstract]
  40. Tokomura, A., Harada, K., Fukuzawa, K., and Tsukatani, H. (1986) Biochim. Biophys. Acta 875, 31-38[Medline] [Order article via Infotrieve]
  41. Smith, D. B., and Corcoran, L. M. (1994) in Current Protocols in Molecular Biology (Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds), pp. 16.7.1-16.7.7, John Wiley & Sons, New York
  42. Post, G. R., Collins, L. R., Kennedy, E. D., Moskowitz, S. A., Aragay, A. M., Goldstein, D., Brown, J. H. (1996) Mol. Biol. Cell 7, 1679-1690[Abstract]
  43. Seilhamer, J. J., Pruzanski, W., Vadas, P., Plant, S., Miller, J. A., Kloss, J., Johnson, L. K. (1989) J. Biol. Chem. 264, 5335-5338[Abstract/Free Full Text]
  44. Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R., Pepinsky, R. B. (1989) J. Biol. Chem. 264, 5768-5775[Abstract/Free Full Text]
  45. Moolenaar, W. H. (1995) J. Biol. Chem. 270, 12949-12952[Free Full Text]
  46. Tigyi, G., Henschen, A., and Miledi, R. (1991) J. Biol. Chem. 266, 20602-20609[Abstract/Free Full Text]
  47. Tigyi, G., and Miledi, R. (1992) J. Biol. Chem. 267, 21360-21367[Abstract/Free Full Text]
  48. Eichholtz, T., Jalink, K., and Moolenaar, W. H. (1993) Biochem. J. 291, 677-680[Medline] [Order article via Infotrieve]
  49. Volwerk, J. J., Pieterson, W. A., and de Haas, G. H. (1974) Biochemistry 13, 1446-1454[Medline] [Order article via Infotrieve]
  50. Lister, M. D., Glaser, K. B., Ulevitch, R. J., Dennis, E. A. (1989) J. Biol. Chem. 264, 8520-8528[Abstract/Free Full Text]
  51. Nicolas, J.-P., Lambeau, G., and Lazdunski, M. (1995) J. Biol. Chem. 270, 28869-28873[Abstract/Free Full Text]
  52. Murakami, M., Hara, N., Kudo, I., and Inonue, K. (1993) J. Immunol. 151, 5675-5684[Abstract/Free Full Text]
  53. Riendau, D., Guay, J., Weech, P. K., Laliberté, F., Yergey, J., Desmarais, S., Perrier, H., Liu, S., Nicoll-Griffith, D., Street, I. P. (1994) J. Biol. Chem. 269, 15619-15624[Abstract/Free Full Text]
  54. Bartoli, F., Lin, H.-K., Gomashchi, F., Gelb, M. H., Jain, M. K., Apitz-Castro, R. (1994) J. Biol. Chem. 269, 15625-15630[Abstract/Free Full Text]
  55. Kudo, I., Murakami, M., Hara, S., and Inonue, K. (1993) Biochim. Biophys. Acta 1170, 217-231[Medline] [Order article via Infotrieve]
  56. Fonteh, A. N., Bass, D. A., Marshall, L. A., Seeds, M., Samet, J. M., Chilton, F. H. (1994) J. Immunol. 152, 5438-5446[Abstract/Free Full Text]
  57. Kishino, J., Kawamoto, K., Ishizaki, J., Verheij, H. M., Ohara, O., Arita, H. (1995) J. Biochem. (Tokyo) 117, 420-424[Abstract]
  58. Tohkin, M., Kishino, J., Ishizaki, J., and Arita, H. (1993) J. Biol. Chem. 268, 2865-2871[Abstract/Free Full Text]
  59. Oka, S., and Arita, H. (1991) J. Biol. Chem. 266, 9956-9960[Abstract/Free Full Text]
  60. Nicolas, J.-P., Lin, Y., Lambeau, G., Ghomashchi, F., Lazdunski, M., and Gelb, M. H. (1997) J. Biol. Chem. 272, 7173-7181[Abstract/Free Full Text]
  61. Ancian, P., Lambeau, G., Mattéi, M.-G., and Lazdunski, M. (1995) J. Biol. Chem. 270, 8963-8970[Abstract/Free Full Text]
  62. Murakami, M., Nakatani, Y., and Kudo, I. (1996) J. Biol. Chem. 271, 30041-30051[Abstract/Free Full Text]
  63. Kramer, R. M., Roberts, E. F., Manetta, J., and Putnam, J. E. (1991) J. Biol. Chem. 266, 5268-5272[Abstract/Free Full Text]
  64. Peppelenbosch, M. P., Qiu, R. G., de Vries-Smits, A. M., Tertoolen, L. G., de Laat, S. W., McCormack, F., Hall, A., Symons, M. H., Bos, J. L. (1995) Cell 81, 849-856[Medline] [Order article via Infotrieve]
  65. Green, J. A., Smith, G. M., Buchta, R., Lee, R., Ho, K. Y., Rajkovic, I. A., Scott, K. F. (1991) Inflammation 15, 355-367[Medline] [Order article via Infotrieve]
  66. Vadas, P., Schouten, B. D., Stefanski, E., Scott, K., and Pruzanski, W. (1993) Crit. Care Med. 21, 1087-1091[Medline] [Order article via Infotrieve]
  67. Vadas, P., Taylor, T. E., Chimsuku, L., Goldring, D., Stefanski, E., Pruzanski, W., and Molyneux, M. E. (1993) Am. J. Trop. Med. Hyg. 49, 455-459[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.