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The Journal of Immunology, 2003, 170: 2093-2099.
Copyright © 2003 by The American Association of Immunologists

Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression 1

Dae-Won Park*, Jae-Ryong Kim*, Seong-Yong Kim*, Jong-Kyung Sonn{ddagger}, Ok-Sun Bang{dagger}, Shin-Sung Kang{dagger}, Jung-Hye Kim* and Suk-Hwan Baek2,*

* Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea; and {dagger} Department of Biology, College of Natural Sciences, and {ddagger} Department of Biology, Teacher’s College, Kyungpook National University, Daegu, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The induction of inducible NO synthase (iNOS) by group IIA phospholipase A2 (PLA2) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA2 up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA2 receptor (sPLA2R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA2 stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA2, Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA2-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA2, and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA2-induced reporter activity of the iNOS promoter. M-type sPLA2R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA2R enhanced group IIA PLA2-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA2 catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA2 induces nitrite production by involving of M-type sPLA2R, which then mediates signal transduction events that lead to PI3K/Akt activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The phospholipase A2 (PLA2)3 family represents a diverse group of enzymes that hydrolyze sn-2 fatty acid from the cell membrane. PLA2 has been implicated in a wide range of cellular responses, including signal transduction, host defense, digestion, and membrane remodeling. Several mammalian cytosolic PLA2 and secretory PLA2 (sPLA2) have been characterized and classified into different families (1, 2, 3, 4). At present, 10 distinct sPLA2 have been identified in mammals and classified into different groups, depending on their primary structures as characterized by the number and position of cysteine residues (5, 6, 7, 8). The sPLA2 have the potential to mediate a wide range of biological activities, for example, they are key components of phospholipid digestion, effectors of antibacterial activity (9), potential regulators of severe illness (10, 11), and cancer markers (12). Although sPLA2 have been studied extensively in mammals, the physiological and pathophysiological functions of these enzymes remain unclear. Therefore, the aim of this study was to investigate the functional diversity of sPLA2.

To date, the biological activity of most sPLA2 has been attributed to their enzymatic capacity to hydrolyze membrane phospholipids and to their role in the production of arachidonic acid and lysophospholipids. However, based on this characteristic, many reports have been difficult to understand (13, 14, 15). Among the sPLA2, group IIA PLA2 is thought to contain enzymes that are key in the pathogenesis of inflammatory diseases, because local and systemic levels are elevated in diseases, such as septic shock, rheumatoid arthritis, and multiple organ failure syndrome (16, 17, 18). However, results on group IIA PLA2-deficient mice suggest that this sPLA2 may not play a pivotal role in the progression and pathogenesis of inflammatory processes, at least in mice (19, 20). Group IIA PLA2 with group IB has been suggested to have a role in cell proliferation and more recently to act as a natural ligand of the sPLA2R on target cells (21). One of the receptors for sPLA2, the M type, displays a high degree of homology with the mannose receptor, a member of the lectin-binding family of receptors that is constitutively expressed on macrophages (22, 23, 24, 25, 26). Moreover, receptor binding on macrophages activates the production of the proinflammatory cytokine IL-6 (27). In addition, macrophages are abundant at sites in which group IIA PLA2 is released in vivo, such as in the synovial fluid or the alveolar space. These observations raised the question as to whether group IIA PLA2 activates macrophages by binding to specific receptors.

The serine/threonine kinases termed Akt or protein kinase B are important regulators of various cellular responses, including glucose metabolism and cell survival (28, 29). The activation of receptor tyrosine kinases and G protein-coupled receptors can lead to both Akt phosphorylation and activation (30, 31). Akt has been identified as a downstream component of phosphatidylinositol 3-kinase (PI3K), and can phosphorylate substrates such as glycogen synthase kinase-3, Bad, and endothelial NO synthase (32, 33, 34). More recently, Akt activation was found to result in the phosphorylation of a number of substrates that have potential importance in macrophage signaling (35). Our hypothesis was that macrophages may be activated by the sPLA2 to induce different mediators, and these mediators are regulated by signal transduction molecules. Little is known of the signal transduction pathway, initiated after occupancy of the sPLA2R. To test these hypotheses, we investigated the ability of group IIA PLA2 to induce inducible NO synthase (iNOS) in macrophages.

In this work, we demonstrate that activation of the macrophage cell line, Raw264.7, by the group IIA PLA2 induces iNOS expression. Moreover, PI3K inhibition by LY294002 results in the down-regulation of iNOS expression, through a mechanism that involves Akt activation, and Akt overexpression enhances the promoter activities of cells cotransfected with plasmids containing a full length of the 5' flanking region of the iNOS gene. Raw264.7 cells express the M-type sPLA2R. In addition, overexpression of M-type sPLA2R in these cells has a profound effect on iNOS expression as induced by group IIA PLA2. These results suggest that Akt plays a role in iNOS expression by mediating the sPLA2R, and might contribute to the understanding of the signal transduction mechanism of group IIA PLA2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Group IIA PLA2 enzyme was purified from the conditioned medium and cell pellets of a HEK293 cell line expressing wild type (WT) or inactive mutant (H48Q) of human group IIA PLA2, by affinity column chromatography, and quantified using a group IIA PLA2 ELISA kit (Cayman Chemical, Ann Arbor, MI). The iNOS-luciferase reporter construct was generously provided by C. Lowenstein (Johns Hopkins Medical Institutions, Baltimore, MD). cDNA of rabbit M-type sPLA2R was obtained from A. Fonteh (Wake Forest University School of Medicine, Winston-Salem, NC). cDNA encoding full length of rabbit M-type sPLA2R, epitope tagged with FLAG peptide, was subcloned into pCR3.1 (Invitrogen, San Diego, CA). The ECL reagent was purchased from NEN Life Science (Boston, MA); RPMI 1640, Lipofectamine 2000, Opti-MEM, and the RT-PCR kit were from Life Technologies (Grand Island, NY); FCS was purchased from HyClone (Logan, UT); rabbit polyclonal iNOS Abs and the HRP-conjugated secondary Abs were from Santa Cruz Biotechnology (Santa Cruz, CA); the phospho-Akt Abs were from New England Biolabs (Beverly, MA); LY294002 was purchased from Biomol (Plymouth Meeting, PA); and p-amino-phenyl-{alpha}-D-mannopyranoside (APMP)-BSA was from Sigma-Aldrich (St. Louis, MO), and dissolved in DMSO before adding to cell cultures. Final DMSO concentrations were 0.1% or less.

Cell culture

The Raw264.7 macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS, grown at 37°C, in 5% CO2 fully humidified air, and subcultured twice weekly. They were then seeded in either a 12-well plate at 1–5 x 105 cells/well or a 6-well plate at 1 x 106 cells/well, and stimulated in the presence of group IIA PLA2 with or without inhibitors for various lengths of time ranging from a few minutes to 24 h.

Stable transfection of group IIA PLA2 and M-type sPLA2R cDNA

Human group IIA PLA2 (WT, H48Q) and rabbit M-type sPLA2R with FLAG were subcloned into the mammalian expression vector pCDNA3.1 or pCR3.1. Lipofectamine 2000 reagent was used to trnasfect sPLA2 or sPLA2R cDNA into HEK293 and Raw264.7 cells, according to the manufacturer’s instructions. One microgram of the plasmid was then mixed with 3 µl of Lipofectamine 2000 in 200 µl of Opti-MEM medium for 20 min, then added to the cells, which had grown to 40–50% confluency in the 6-well plate. After incubation for 5 h, the medium was replaced with fresh culture medium. To obtain stable transfectants, the cells transfected with the cDNA were cloned by serial dilution in 96-well plate in a culture medium containing 1000 µg/ml of G418. The subculturing was continued for 4 wk, and then wells representing a single colony were selected and the expression was confirmed using its mRNA level as determined by Northern blot analysis or RT-PCR.

NO assay

NO synthesis was determined by assaying the culture supernatant for nitrite, the stable reaction product of NO. Briefly, 100 µl of the culture supernatant was allowed to react with 100 µl of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) at room temperature for 10 min. The OD of the assay sample was measured spectrophotometrically at a wavelength of 570 nm. Fresh culture medium was used as a blank in all experiments. Nitrite concentration was calculated from a standard curve prepared using NaNO2 under the same assay conditions.

PI3K assay

Raw264.7 cells were seeded in 35-mm dishes and cultured overnight before they were serum starved for 24 h and treated with or without group IIA PLA2 for the indicated times. Whole cell lysates were prepared in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 µM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail. For immunoprecipitation, 500 µg of total protein was incubated with phosphotyrosine Ab (4G10) for 4 h, and then with protein A-agarose for 2 h at 4°C. After seven washes in lysis buffer, the immunocomplexes were resolved by SDS-PAGE, and transferred to nitrocellulose for subsequent immunoblot analysis.

PLA2 activity assay

PLA2 activities of WT and mutant were measured as acylhydrolysis of 1-palmitoyl-2-[1-14C]linoleoyl L-3-phosphatidylethanolamine. The samples were incubated with the enzyme and substrate (total 20,000 cpm) for 30 min at 37°C. Results are calculated as cpm free fatty acid hydrolyzed.

Transfection and luciferase activity assay

Raw264.7 cells were plated to a density of 2 x 105 cells per 12-well plate. The iNOS promoter gene ligated to the luciferase gene was transfected into the cells using Lipofectamine 2000. After transfection, the cells were placed in complete medium for 16 h, and group IIA PLA2 was added for an additional 24 h. The cells were then harvested, washed, and lysed with reporter lysis buffer (Promega, Madison, WI). The lysed cells were briefly centrifuged, and the relative reporter induction strength was calculated by measuring the luciferase activity of the supernatant using luciferase assay reagent (Promega). The transfection efficiency was normalized by cotransfecting the cells with 0.5 µg of an expression plasmid containing a CMV promoter-driven {beta}-galactosidase reporter.

Western blot analysis

Raw264.7 cells were plated in 6-well plate (1 x 106 cells/well) and treated with group IIA PLA2 for 24 h. The cells were washed with cold PBS, scraped off, and pelleted at 700 x g at 4°C. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail), the preparation was cleared by centrifugation, and the supernatant was retained as a whole cell lysate. Protein (20 µg) was separated by 8% reducing SDS-PAGE and immunoblotted in 20% methanol, 25 mM Tris, and 192 mM glycine to a nitrocellulose membrane, which was then blocked with 5% nonfat dry milk in TTBS (25 mM Tris-HCl, 150 mM NaCl, and 0.2% Tween 20) and subsequently incubated with the indicated Abs for 4 h. The membrane was then washed and incubated for 1 h with secondary Abs conjugated to HRP, rewashed, and developed using an ECL system.

RT-PCR

Raw264.7 cells (2 x 106 cells) were cultured, harvested, and washed three times with PBS containing 2% BSA, and the RNA was isolated using an RNeasy kit (Qiagen, Chatsworth, CA). A modified RT-PCR technique was used to determine the receptor mRNA level. Briefly, the total RNA was reverse transcribed into cDNA by RT-PCR. Oligonucleotide primers for the PCR were designed according to the published sequence for the mouse M-type sPLA2R (sense, ATT ATC CAG AGC GAG AGC CT; antisense, TCT TGC CAC CAC TGT GTT GT) and for the rabbit M-type sPLA2R with FLAG (sense, ATG GAC TAC AAA GAC GAT GAC GAC ATG CTG CTG TCC CTG CTG; antisense, ACT ATC TGA CAT ATA GGA TAT CCA CTT AT). The PCR conditions used for M-type sPLA2R production were 30 cycles of denaturation (95°C/l min), annealing (61°C/30 s), and extension (72°C/1 min) in the presence of 2.5 mg MgCl2, followed by a final 20-min extension at 72°C. The oligonucleotide primers for {beta}-actin were used as a control for RT-PCR. The PCR products were separated by electrophoresis through a 1% agarose gel containing ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Group IIA PLA2 stimulates iNOS expression and nitrite production

Cells overexpressing this gene in HEK293 cells were constructed using a mammalian expression system to investigate the biological function of group IIA PLA2. After transfecting the cDNA of group IIA PLA2, affected G418 cells were selected. A total of 14 G418-resistant clones were obtained. After culturing and harvesting, sPLA2 expression was confirmed by Northern blot analysis and PLA2 activity (data not shown). sPLA2 proteins were purified from the overexpressing cells by affinity column chromatography, quantified using an ELISA kit. Endotoxins were removed using Vivapure mini columns (VivaScience, Goettingen, Germany), and the endotoxin content was determined by using a Kinetic Quantitative Chromogenic Limulus Amoebocyte Lysate kit (BioWhittaker, Walkersville, MD). To examine the iNOS expressional response to sPLA2, Raw264.7 cells were stimulated with increasing concentrations of sPLA2, and nitrite production and iNOS expression were determined. Basal nitrite production occurred at very low levels; however, sPLA2 stimulated nitrite production in a dose-dependent manner, and this increased level of nitrite production correlated with iNOS protein expression (Fig. 1A). When the cells were treated with sPLA2 for various times (2–24 h), both iNOS expression and nitrite production increased significantly in a time-dependent manner (Fig. 1B).



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  		FIGURE 1. Group IIA PLA2 induced iNOS expression and nitrite production in Raw264.7 cells. The cells were stimulated with the indicated sPLA2 doses for 24 h (A), treated with sPLA2 (2 µg/ml), and harvested at the indicated time points (B). Nitrite production was measured in the culture supernatant, as described in Materials and Methods. The cell lysates were resolved by SDS-PAGE and probed using iNOS Abs. The values for the nitrite concentrations are represented as averages ± SE of three independent experiments. The iNOS protein levels are representative of three independent experiments.

 
Akt mediates iNOS induction by group IIA PLA2

The Raw264.7 cells were treated with the sPLA2 for various times and used to prepare whole cell extracts for Western blot analysis of Akt phosphorylation. Abs against phosphorylated Akt (serine 473) were used to immunoblot Akt phosphorylation. As shown in Fig. 2A, a rapid and transient increase in Akt phosphorylation was observed after sPLA2 treatment, within 1 min enzyme phosphorylation increased, and maximum phosphorylation was observed within 5 min of stimulation. The Akt phosphorylation, induced by sPLA2, was abolished by LY294002, a specific PI3K inhibitor (Fig. 2B). Next, we examined whether PI3K, an upstream target of Akt, is activated in response to group IIA PLA2 stimulation. Cells were treated with group IIA PLA2 for various times, and whole cell extracts were prepared for PI3K immunoprecipitation. As shown in Fig. 2C, after treatment of group IIA PLA2, PI3K activity showed a rapid, but transient increase. Maximum PI3K activation was observed within 1 min of stimulation, and this activation was inhibited by LY294002 (Fig. 2D). To determine whether iNOS induction occurred downstream of PI3K/Akt activation, Raw264.7 cells were pretreated with LY294002, and then stimulated with various doses of sPLA2. Nitrite production in the culture medium and iNOS expression in the cell lysates were then determined. As shown in Fig. 3, LY294002 strongly inhibited group IIA PLA2-induced iNOS expression and nitrite production.



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  		FIGURE 2. sPLA2-induced phosphorylation of Akt is linked to PI3K activation. A, The cells were treated with sPLA2 (2 µg/ml) for the indicated times, and B, pretreated with or without 25 µM of LY294002 for 30 min and then stimulated with sPLA2. The lysates were analyzed by Western blot with Abs to phospho-Akt and Akt. The two protein levels shown are representative of three independent experiments. C, The cells were treated with sPLA2 for the indicated times, and D, with sPLA2 for 1 min with or without LY294002 (25 µM). Total protein (500 µg) was immunoprecipitated (IP) with 4G10, followed by immunoblot (IB) with anti-p85 Ab. Whole cell extracts (30 µg) were subjected to Western blot analysis to determine same protein levels.

 


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  		FIGURE 3. sPLA2-induced iNOS expression and nitrite production are linked to PI3K/Akt activation. Cells were treated with the indicated concentrations of sPLA2 in the presence or absence of LY294002 (25 µM) for 16 h. Nitrite production was measured in the culture supernatant. Whole cell lysates were obtained, and Western blot analysis was performed using Ab specific for iNOS. Values of the nitrite concentrations are represented as averages ± SE of three independent experiments. The iNOS protein levels are representative of three independent experiments.

 
Group IIA PLA2 stimulates iNOS promoter, which is inhibited by LY294002

To determine whether the stimulation of nitrite production by the sPLA2-activated macrophages was due to an increase in the transcriptional activation of iNOS, promoter-luciferase fusion constructs containing full lengths of the 5' flanking sequences of the iNOS gene were obtained. Luciferase activities were measured in Raw264.7 cells that were transiently transfected with these constructs and treated with sPLA2. These results were then compared with those of the untreated control cells. As shown in Fig. 4A, cells transfected with the control vector showed a basal level of luciferase activity. However, a strong increase in the level of luciferase activity was consistently observed (40-fold) after the sPLA2 stimulation, suggesting that the increase in nitrite production observed in these cells is due to the activation of the iNOS transcripts. To obtain mechanistic information on the inhibition of iNOS expression by LY294002, the effect of LY294002 on iNOS promoter activity was examined. After 16-h transfection with iNOS promoter DNA, Raw264.7 cells were pretreated with 25 µM of LY294002 for 30 min and then stimulated with the sPLA2 for 24 h before performing a luciferase assay on the lysed cell extracts. LY294002 alone did not affect the basal luciferase activity. However, pretreatment with LY294002 strongly inhibited sPLA2-induced iNOS reporter activity (Fig. 4B). To further confirm the role of Akt, the cells were cotransfected with iNOS promoter DNA and either a plasmid-encoding control vector (pEGFP) or the wild form of Akt. Consistently, these plasmid-encoding control vectors had only minimal effects on the reporter activity of cells stimulated with the sPLA2. However, cotransfection of plasmid-encoding iNOS promoter with a wild form of Akt had profound effects on the reporter activity (Fig. 5), which suggests that Akt might be involved in the signaling pathway triggered by sPLA2.



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  		FIGURE 4. Stimulation of iNOS promoter by sPLA2 and inhibition by LY294002. A, Cells were transiently transfected with the control vector or the cDNA of iNOS promoter and then stimulated with sPLA2 for 24 h. B, Cells were transfected with the cDNA of iNOS promoter and then treated with the sPLA2 in the presence or absence of 25 µM of LY294002. In each case, the luciferase activity was measured, as described in Materials and Methods. The results are expressed relative to the luciferase activity detected in nontreated cells. Luciferase activities are represented as averages ± SE of five independent experiments.

 


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  		FIGURE 5. Akt overexpression increases sPLA2-induced iNOS promoter activity in Raw264.7 cells. Cells were transiently cotransfected with the cDNA of iNOS promoter and either a control vector (pEGFP) or WT Akt. Cells were incubated for 16 h, then stimulated with the sPLA2 for 24 h. Whole cell lysates were obtained, and Western blot analysis was performed using Ab specific for Akt. Luciferase activity was measured, as described in Materials and Methods. The results are expressed relative to the luciferase activity in nontreated cells. Luciferase activities are represented as averages ± SE from five independent experiments.

 
M-type sPLA2R is linked to nitrite production by sPLA2

To determine whether sPLA2-mediated nitrite production is dependent upon its cell surface receptor, we investigated sPLA2R expression in Raw264.7 cells. Total RNA was isolated from cultured cells, and RT-PCR analysis was conducted to identify the presence of the M-type sPLA2R. The results shown in Fig. 6A demonstrate that the M-type sPLA2R is highly expressed by Raw264.7 cells. This receptor was also detected in the bone marrow-derived mast cells of BALB/C mice and in NIH3T3 cells, using the primer for mouse M-type sPLA2R reported previously (36, 37). To examine the nitrite production and iNOS expression requirement for sPLA2Rs, cells were cotransfected with the cDNA of the iNOS promoter either as a control vector (pCR3.1) or M-type sPLA2R epitope tagged with FLAG peptide. After transfection, both the vector and receptor-expressing cells were stimulated with sPLA2, and as shown in Fig. 6B, sPLA2 induced promoter activity in a dose-dependent manner in the vector-transfected cells. However, sPLA2 markedly increased the promoter activities of cells overexpressing M-type sPLA2R. To demonstrate that these events are tightly linked to the PI3K/Akt pathway, we treated cells with the PI3K inhibitor LY294002 and evaluated the promoter activity of iNOS. sPLA2-induced promoter activities were inhibited by LY294002 under both conditions. To study the role of M-type sPLA2R in nitrite production, we transfected with either a vector or M-type sPLA2R cDNA and stimulated with group IIA PLA2. As shown in Fig. 6C, transfection of the M-type sPLA2R had profound effects on nitrite production and iNOS expression compared with that of the vector-transfected cells. In addition, sPLA2-induced nitrite production and iNOS expression were strongly inhibited by LY294002. To confirm the effect of receptor-mediated induction of iNOS expression, we established the stable Raw264.7 cells with rabbit M-type sPLA2R cDNA. Rabbit M-type sPLA2R did not detect in Raw264.7 cells by RT-PCR. By cDNA transfection, the cells expressing high levels of receptor were obtained and expanded into cell lines. After confirming the receptor expression, we tested whether the stimulatory effect of group IIA PLA2 could be reproduced by using Raw264.7 cells expressing receptor. As shown in Fig. 7A, sPLA2 highly induced nitrite production and iNOS expression in cells expressing sPLA2R compared with that of the vector-transfected cells. In addition, LY294002 totally blocked expression of iNOS induced by sPLA2. These results suggest that the M-type sPLA2R might be involved in nitrite production mediated by the PI3K/Akt pathway. To determine whether catalytic activity is required to induce NO production through the sPLA2R, the effects of a noncatalytic ligand of sPLA2R, APMP-BSA, were examined on the sPLA2-induced nitrite production. The APMP-BSA alone did not affect nitrite production. However, APMP-BSA significantly inhibited sPLA2-induced iNOS expression and nitrite production (Fig. 7B). Moreover, to assess the contribution made by the activity of sPLA2 to its unique ability to induce iNOS, we generated a mutant IIA PLA2, in which His-48, the residue essential for the hydrolytic activity of PLA2, was mutated to Gln. The active site mutant H48Q was produced by site-directed mutagenesis; the resulting mutant was sequenced to confirm the presence of the mutation. A pool of cells expressing the H48Q mutant was obtained using the same method as that described for the HEK293 cells, and the expressions and activities of the mutant PLA2 were confirmed. As shown in Fig. 8, H48Q PLA2 had less than 10% of the activity of the WT, and these mutants had only weak effects on iNOS expression and nitrite production compared with the WT.



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  		FIGURE 6. Overexpression of M-type sPLA2R increased the promoter activity and protein expression of iNOS. A, Cells were cultured and harvested at a confluency of 80%. Total RNA was extracted, and RT-PCR was performed using specific primers for the mouse M-type sPLA2R. B, Cells were transiently cotransfected with the cDNA of iNOS promoter and either a control vector (pCR3.1) or rabbit M-type sPLA2R epitope tagged with FLAG. Luciferase activity was measured, as described in Materials and Methods. The results are expressed relative to the luciferase activity of the nontreated cells. Luciferase activities are represented as averages ± SE of four independent experiments. C, Cells were transiently transfected with either a control vector (pCR3.1) or rabbit M-type sPLA2R epitope tagged with FLAG. Approximately 16 h transfection, cells were stimulated with the various concentrations of sPLA2 for 24 h in the absence or presence of LY294002. Expression of M-type sPLA2R, as assessed by RT-PCR. Nitrite production was measured in the culture supernatant. Whole cell lysates were obtained, and Western blot analysis was performed using Ab specific to iNOS. iNOS and sPLA2R levels are representative of three independent experiments.

 


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  		FIGURE 7. M-type sPLA2R activates sPLA2-induced iNOS expression. A, Raw264.7 cells were transfected with either a control vector (pCR3.1) or rabbit M-type sPLA2R, as indicated, and established stable transfectants. Control vector or rabbit M-type sPLA2R transfectants were stimulated with the various concentrations of sPLA2 for 24 h in the absence or presence of LY294002 (25 µM). The top panel shows the expression of M-type sPLA2R in both vector and sPLA2R stable transfectants. B, Raw264.7 cells were stimulated with sPLA2 in the presence of the indicated doses of APMP-BSA for 24 h. Nitrite production was measured in the culture supernatant. Whole cell lysates were obtained, and Western blot analysis was performed using Ab specific to iNOS. iNOS levels are representative of three independent experiments.

 


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  		FIGURE 8. Comparison of iNOS regulation in the WT PLA2 and in the H48Q mutant. A, WT PLA2 and mutant H48Q PLA2 were purified from HEK 293 cell transfections, and PLA2 activities were measured using 1-palmitoyl-2-[1-14C]linoleoyl L-3-phosphatidylethanolamine as substrates. Expression of wild and mutated sPLA2 in HEK293 transformants as assessed by RT-PCR. B, Raw264.7 cells were stimulated with wild and mutated sPLA2 for 24 h and harvested. Nitrite production was measured in the culture supernatant, as described in Materials and Methods. The cell lysates were resolved by SDS-PAGE and probed using iNOS Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that Akt is activated in macrophages by sPLA2 and that Akt activation is required for iNOS induction and nitrite production. Treatment with LY294002, a PI3K inhibitor, attenuated sPLA2-induced Akt activation, iNOS promoter activity, and protein expression. Cells expressing WT Akt exhibited more potent activation of iNOS promoter by sPLA2. Moreover, macrophages transfected with the M-type sPLA2R showed increased iNOS reporter activity and protein expression upon sPLA2 stimulation, which suggests that the sPLA2-mediated receptor activation stimulates iNOS induction through the PI3K/Akt cascade.

Although the NO produced by iNOS accounts for the bactericidal and tumoricidal properties of macrophages, NO is also of particular importance in the pathophysiology of various inflammatory diseases, including ischemia, septic shock, and the production of proinflammatory cytokines (38, 39, 40). Moreover, massive production of NO can have detrimental effects on the host organism. For this reason, the mechanism of macrophage activation has attracted considerable interest. The present study shows that in response to sPLA2, Raw264.7 cells rapidly stimulate Akt phosphorylation, and that this activity peaks at 5–10 min and then quickly subsides, thus indicating that Akt activation, like many other cellular responses, adapts rapidly. Akt activation in cells requires the functional involvement of the closely related PI3K, which is activated in response to a variety of extracellular signals (30, 31, 32, 33). The role of PI3K in Akt activation is supported by the results of our study, which demonstrated that the PI3K inhibitor, LY294002, effectively blocks sPLA2 stimulation. Moreover, transfection and inhibition studies strongly suggest the direct involvement of Akt in iNOS induction. The treatment of Raw264.7 cells with sPLA2 resulted in Akt phosphorylation via PI3K, which led to iNOS expression and resulted in the up-regulation of nitrite production. There have been no reports of sPLA2-induced activation of Akt downstream of PI3K. Akt is a serine/threonine kinase that has significant homology with protein kinase C and protein kinase A (41, 42), and has been reported to mediate the effects of insulin, growth factors, cytokines, and various other factors. The downstream effects of Akt include the inhibition of a number of proapoptotic factors, and the activation of I-{kappa}B{alpha} kinase and endothelial NO synthase (34, 43, 44). In fact, some reports suggest that PI3K is the regulator of iNOS expression by LPS. However, these results are controversial because other reports have suggested that PI3K plays a negative role in LPS-induced iNOS expression (45, 46), while Salh et al. (47) demonstrated that LY294002 inhibited LPS-induced NO production in the macrophage cells. Although the evaluation of the relationship between sPLA2 and macrophage activation is beyond the scope of this study, it is possible that the sPLA2 activation of Akt is related to iNOS induction during macrophage activation.

The growth-promoting effects of sPLA2 have been observed in a variety of murine and human cells, including Swiss 3T3 cells, NIH3T3 cells, synovial cells, and chondrocytes (37, 48, 49, 50). Group IB PLA2 activated mitogen-activated protein kinase (MAPK) cascade, and a selective MAPK kinase inhibitor blocked sPLA2-induced cell proliferation, suggesting that the MAPK cascade is essential for group IB PLA2-induced cell proliferation (51). Although much effort has been made to identify the intracellular signaling pathway triggered by sPLA2, the signal transduction mechanism and the biological function are still unclear. In various types of cells, the activation of eicosanoid formation by group IB PLA2 has been identified; this response was mediated by sPLA2R. For example, mouse osteoblastic MC3T3-E1 cells enhanced PGE2 production after stimulation with porcine group IB PLA2. During this activation process, the cyclooxygenase-2 (COX-2) mRNA level increased in parallel with elevated PGE2 production, indicating that the main pathway in the group IB PLA2-induced production of PGE2 involves the induction of the COX-2 gene (52). Bidgood et al. (53) reported that group IIA PLA2 up-regulates COX-2 and amplifies cytokine-mediated PGE2 production in human rheumatoid synoviocytes. We also observed that the group IIA PLA2 stimulates both COX-2 expression and PGE2 formation in Raw264.7 cells.

NO production in many cells is due to the activation of various signal molecules, and this process is often accompanied by the phosphorylation of MAPKs (54). MAPKs initially appear to be the enzymes primarily responsible for iNOS regulation. However, other kinase pathways have also been shown to regulate iNOS expression (47, 55). Important questions arise from these studies concerning the signal transduction mechanism of this type of iNOS induction. The current study has focused on signal transduction events that are closely associated with iNOS regulation. Because Akt is a PI3K downstream and can be potently activated by PI3K, it is important to determine whether Akt might be an integral part of sPLA2 signaling. This shows that during sPLA2R occupancy, Akt is transiently phosphorylated. In fact, in stimulated macrophages, inhibition studies have shown that both MAPKs and Akt are responsible for iNOS regulation. Incubation of macrophages with a selective PI3K inhibitor or MAPK kinase inhibitor significantly blocked iNOS expression and nitrite production in response to sPLA2. However, LY294002 did not affect MAPK phosphorylation by sPLA2 (data not shown), which suggests that multiple signal transduction pathways regulate group IIA PLA2-induced iNOS expression in activated macrophages.

To date, much of the biological activity of sPLA2 has been attributed to its enzymatic capacities to hydrolyze membrane phospholipids. However, pancreatic sPLA2 has been shown to promote cell proliferation, cell migration, hormone release, and acute lung injury. In addition, M-type sPLA2R has been suggested to play a major role in these effects (21, 24, 25, 26). The present study suggests that sPLA2R occupancy is linked to iNOS induction. In addition, M-type sPLA2R expression was confirmed in Raw264.7 macrophage cell, which implies that binding alone can produce nitrite in cells. Over the past decade, sPLA2Rs have been described on the basis of their binding properties in the tissue or cell. A cloned sPLA2R, called the M-type receptor, has homology with the macrophage mannose receptor and the DEC-205 of dendrites, suggesting that these proteins may belong to a new class of receptors that possess agonist recognition properties. The 180-kDa M-type sPLA2R was initially identified using snake venom sPLA2. A first clue to the physiological function of the M-type receptor was provided when it was observed that the mammalian pancreatic group IB PLA2, but not the inflammatory group IIA PLA2, was a ligand of the M-type receptor. However, other binding experiments to rabbit M-type receptor suggested that both group IB and group IIA PLA2 may be natural endogenous ligands of this receptor (21). The current study shows that transient or stable overexpression of sPLA2R is increased iNOS expression and nitrite production. In addition, APMP-BSA, a noncatalytic ligand of the sPLA2R, significantly suppressed the nitrite production and iNOS expression by sPLA2. However, inactive sPLA2 (H48Q) had a very weak effect on iNOS expression (Fig. 8). Furthermore, sPLA2 inhibitors, such as p-bromophenacyl bromide, 12-epi-scalaradial, and aristolochic acid, attenuated sPLA2-induced nitrite production (data not shown). The failure of inactivated sPLA2 to increase nitrite production may be due to its inability to bind with sPLA2R in cells. Together these data suggest that the binding of sPLA2 to a M-type receptor requires sPLA2 activity. Further studies will be necessary to determine relationship between sPLA2 activity and receptor binding.

Overall, our results suggest that the physiologic roles of group IIA PLA2 are not only mediated by hydrolytic effects, but also by their capacity to bind to cell surface receptors. Upon receptor binding, group IIA PLA2 induces the PI3K/Akt signal pathway that leads to iNOS expression and nitrite production.


   Acknowledgments
 
We thank Dr. C. Lowenstein for providing iNOS-luciferase reporter construct, and Dr. A. Fonteh for the gift of cDNA of rabbit M-type sPLA2R.


   Footnotes
 
1 This work was supported by Korea Research Foundation Grant (KRF-2000-DP0352). Back

2 Address correspondence and reprint requests to Dr. Suk-Hwan Baek, Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, 317-1 Daemyung-5 Dong, Nam-Gu, Daegu 705-035, South Korea. E-mail address: sbaek{at}med.yu.ac.kr Back

3 Abbreviations used in this paper: PLA2, phospholipase A2; APMP, p-amino-phenyl-{alpha}-D-mannopyranoside; COX-2, cyclooxygenase-2; iNOS, inducible NO synthase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; sPLA2, secretory PLA2; WT, wild type. Back

Received for publication July 13, 2002. Accepted for publication December 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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