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Originally published In Press as doi:10.1074/jbc.M705709200 on November 20, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3077-3087, February 8, 2008
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Activation of Cytosolic Phospholipase A2{alpha} through Nitric Oxide-induced S-Nitrosylation

INVOLVEMENT OF INDUCIBLE NITRIC-OXIDE SYNTHASE AND CYCLOOXYGENASE-2*

Lihong Xu, Chang Han, Kyu Lim, and Tong Wu1

From the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Received for publication, July 11, 2007 , and in revised form, November 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytosolic phospholipase A2{alpha} (cPLA2{alpha}) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E2 (PGE2), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA2{alpha} protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA2{alpha} S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA2{alpha} activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA2{alpha} protein induced S-nitrosylation of cPLA2{alpha} in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA2{alpha} activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA2{alpha} in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA2{alpha} is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA2{alpha} S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE2 production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA2{alpha} S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA2{alpha}, and COX-2. Since COX-2 enhances the formation of cPLA2{alpha}-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA2{alpha} S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA2{alpha}. These findings disclose a novel link among cPLA2{alpha}, iNOS, and COX-2, which form a multiprotein complex leading to cPLA2{alpha} S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE2 production and prevent inflammation and carcinogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytosolic phospholipase A2{alpha}(cPLA2{alpha})2 plays critical roles in a variety of physiological and pathophysiological conditions through generating a cascade of bioactive lipid mediators. The cPLA2{alpha} enzyme catalyzes hydrolysis of membrane glycerophospholipids to selectively release free arachidonic acid (AA), which then acts as the substrate for cyclooxygenase (COX) to produce prostaglandins (PGs), for lipoxygenase to synthesize leukotrienes and hydroxyeicosatetraenoic acids, and for cytochrome P450s to generate epoxyeicosatrienoic acids (1–6). These lipid products function as local hormones through binding to their cellular receptors in autocrine or paracrine fashions or serve as intracellular second messengers to mediate a myriad of physiological and pathophysiological functions, such as inflammation, cell proliferation, and carcinogenesis. In normal cells, the activity of cPLA2{alpha} is tightly regulated by several intracellular signaling events, including intracellular calcium influx and enzyme phosphorylation (1–6). In disease processes, dysregulation of cPLA2{alpha} activity and imbalance of AA release has been implicated in the pathogenesis of inflammatory and neoplastic disorders, such as allergic reactions, arthritis, acute lung injury, autoimmune disease, and several types of cancers, some of which are accompanied by PGE2 accumulation (7–20).

Recent evidence suggests that functional coupling between cPLA2{alpha} and COX-2 represents an important mechanism for prostaglandin synthesis (5, 21–24). COX-1 and COX-2 have a similar Km for arachidonic acid, but COX-2 metabolizes the AA cleaved by activated cPLA2{alpha} (5, 21, 22). The exact molecular mechanism for the coupling between cPLA2{alpha} and COX-2 remains to be further defined, although possible explanations include co-localization of the enzymes in certain cellular compartments and/or involvement of unknown binding proteins (5).

The biosynthesis of prostaglandins is regulated by nitric oxide (NO) in various cell types (25). This phenomenon was first reported by Salvemini et al. (26), who showed that NO activated cyclooxygenase and enhanced PG synthesis in the mouse macrophage cell line RAW264.7. Since then, this observation was confirmed and extended by other investigators in various cellular systems and animal models (for a review, see Ref. 25). Therefore, COX enzymes may represent important endogenous targets for modulating the multifaceted roles of NO. Kim et al. (27) reported that iNOS-derived NO S-nitrosylates COX-2 and thus enhances COX-2 catalytic activity in macrophages, which provides mechanistic explanation for NO-induced COX-2 activation. Recently, some NO donors combined with several nonsteroidal anti-inflammatory drugs (e.g. nitroaspirin and nitroflurbiprofen) have been synthesized and used in the treatment of inflammatory and noninflammatory disorders, suggesting the possible beneficial effect of drugs acting on NO and PGs simultaneously. On the other hand, fatty acid has been shown to act as the storage of NO in lipid bilayers and micelles and as the regulator of NO release (28). In human blood plasma and urine, nitrofatty acids are abundantly present (29, 30), and nitrolinoleic acid is a potent endogenous ligand for peroxisome proliferator-activated receptor-{gamma} (31). Despite these achievements, however, the potential effect of NO on the regulation of PLA2 has not been studied.

This study was designed to investigate whether nitric oxide signaling might modulate cPLA2{alpha} activation. Our data indicate that cPLA2{alpha} is S-nitrosylated and activated by iNOS-derived NO in human epithelial cells. This effect is mediated by Cys-152 of cPLA2{alpha}. Thus, S-nitrosylation of cPLA2{alpha} may represent an important mechanism for regulation of AA release and eicosanoid biosynthesis. Furthermore, we show that COX-2 facilitates the association between iNOS and cPLA2{alpha}, thereby dramatically enhancing iNOS/NO-induced cPLA2{alpha} S-nitrosylation and PGE2 synthesis. These results reveal a novel iNOS-mediated functional coupling between cPLA2{alpha} and COX-2 for efficient PG synthesis in human cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The prostaglandin E2 Biotrak Enzymeimmunoassay System (catalog number RPN222) was purchased from GE Healthcare. [5,6,8,9,11,12,14,15-3H(N)]Arachidonic acid (NET-298) was from PerkinElmer Life Sciences. Recombinant human IL-1β and TNF-{alpha} were from R&D Systems (Minneapolis, MN). Recombinant human interferon-{gamma}, L-arginine, S-methyl methanethiosulfonate, CHAPS solution, Triton X-100, sodium L-ascorbate, and neocuproine were from Sigma. S-Nitrosoglutathione (GSNO), 1400W, NOC-18, anti-human COX-2 antibody, and the cPLA2 assay kit (catalog number 765021) were purchased from Cayman Chemical (Ann Arbor, MI). N Gmonomethyl-L-arginine and NS-398 were from EMD Biosciences (San Diego, CA). N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide and immobilized Streptavidin were from Pierce (Rockford, IL). Micro Bio-Spin Chromatography Column was from Bio-Rad. Anti-human iNOS and cPLA2{alpha} antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). The cell culture medium and serum were from Invitrogen.

Cell Culture, Transfection, and Transduction—Four types of human epithelial cells were utilized in this study (liver epithelia-derived SG231 and CCLP1, lung epithelia-derived A549, and kidney epithelia-derived HEK293). SG231 cells were cultured in {alpha}-minimal essential medium supplemented with 10% fetal bovine serum (FBS) as we described (32, 33). CCLP1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (32, 33). A549 cells (obtained from ATCC) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. HEK293 cells (obtained from ATCC) were cultured in Eagle's minimum essential medium containing 10% FBS. All of the cells were cultured at 37 °C in a humidified CO2 incubator. For overexpression of iNOS in these cells, the iNOS virus or control Y5 virus (provided by the University of Pittsburgh Vector Core Facility) at different titers were added into the culture medium without FBS for 2 h, followed by 5% FBS overnight. The cPLA2{alpha} expression plasmid was transiently transfected into the cells with low expression of endogenous cPLA2{alpha} (CCLP and HEK293). For this purpose, the cells were seeded on the 6-well plate with 10% FBS the day before transfection. On the following day, the cells in each well (80% confluence) were transfected with 1 µg of cPLA2{alpha} or control MT2 plasmid using Lipofectamine Plus reagent (6 µl of Plus reagent, 4 µl of Lipofectamine) in serum-free medium. For CCLP1 cells, the transfection medium was replaced with culture medium containing 5% FBS 3 h later. For HEK293 cells, the transfection reagents were removed after 2 h and replaced with regular medium with 5% FBS. After an additional 16-h incubation, the culture medium was collected for PGE2 production assay, whereas the cells were washed twice with cold PBS and subjected to further analysis as described under "Results."

Cytokine Treatment—SG231, A549, and HEK293 cells were seeded onto 6-well plates. After 1 day in culture, the cells at ~70–80% confluence were incubated with vehicle or cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) in 0.5% FBS medium for 24 h. For adenovirus infection plus cytokine mixture treatment, the cells were incubated with vector for 2 h in 0.5% FBS medium, followed by incubation with cytokine mixture in 0.5% FBS medium overnight.

In Vitro cPLA2 Activity Assay—2-Deoxy-2-thioarachidonoylphosphatidylcholine was used as the substrate to measure cPLA2 activity in vitro. The assay was performed by using the cPLA2 assay kit purchased from Cayman Chemical (catalog number 765021). The secretary PLA2 and calcium-independent PLA2 were excluded or deactivated from the whole cell lysate to improve the assay specificity. All procedures were performed according to the manufacturer's instructions with minor modifications. In brief, after transfection with plasmids or infection by adenovirus vector or treatment with different reagents, the cells were scraped down from plates and homogenized in Hepes-EDTA buffer. The homogenates were then centrifuged to remove cell debris at 14,000 rpm for 20 min. To minimize contamination of secretary PLA2 and calcium-independent PLA2, supernatant was concentrated by an Ambion Y30 filter, followed by incubation for 20 min with bromoenol lactone, a calcium-independent PLA2 inhibitor. 10 µl of cell lysate was finally subjected to the assay in the 96-well mode, and the OD value was measured at 414 nm.

[3H]Arachidonic Acid Release—The cells seeded on 6-well plates were treated with various reagents or infected with adenovirus in the presence or absence of cytokine mixture for 24 h. [5,6,8,9,11,12,14,15-3H]arachidonic acid (0.5 µCi/ml) was simultaneously added into culture medium containing 1% serum and left for incorporation into cell membranes overnight. On the next day, the culture media were removed, and the cells were rinsed five times with medium, followed by incubation with medium containing 0.5% serum for 1–2 h. The culture medium was then collected and centrifuged at 14,000 rpm for 20 min to remove suspension cells. 3H activity was measured using a liquid scintillation counter.

PGE2 Production Assay—The cells were treated with different reagents or infected with adenovirus for 24 h. The culture medium was then collected and centrifuged at 14,000 rpm for 20 min to exclude suspension cells. 50 µl of supernatant, with triplicate wells for each sample, was utilized for enzyme immunoassay. Triplicate blank wells and nonspecific binding wells were included to minimize the interference of background color. The binding of PGE2 with antibody lasted for 2 h at room temperature, which was followed by five rinses with wash buffer to maximally eliminate unbound or nonspecifically bound antibody. 150 µl of enzyme substrate was added into each well for 30 min, and the OD value was measured at 630 nm using an enzyme-linked immunosorbent assay reader. The actual PGE2 concentration was calculated according to the standard curve ranging from 0 to 320 pg/50 µl. The samples with too high concentration of PGE2 were diluted appropriately for further assay.

Detection of S-Nitrosylation of cPLA2{alpha}—A biotin switch assay was utilized to measure the S-nitrosylation of cPLA2{alpha} according to the previously described methods (34, 35) with modifications. For this assay, the unnitrosylated thiol groups are first blocked by S-methyl methanethiosulfonate; the targeted nitrothiols are then reduced to free thiols by ascorbate; the free thiols are subsequently labeled with biotin through a synthesized chemical, N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide; finally, the biotinylated protein is immunoprecipitated by streptavidin beads and subjected to visualize by regular Western blotting. Specifically, the cells after cytokine treatment or transfection (as specified) were scraped down from plates in PBS buffer and centrifuged to obtain cell pellets, which were then homogenized in 200 µl of HEN buffer (250 mM Hepes-NaOH, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine). The mixture was centrifuged at 2000 x g for 10 min at 4 °C, and supernatant was recovered as crude cell lysate. CHAPS (final concentration of 0.4%) solution was added to further dissolve membrane proteins. For measurement of S-nitrosylation induced by NO donor, isolated cell lysate was incubated with GSNO (20 µM) for 20 min at room temperature, followed by removal of NO donor through P-6 column (from Bio-Rad). For measurement of S-nitrosylation in cells related to iNOS overexpression, cell lysate was directly blocked in 600 µl of blocking buffer (HEN buffer, 2.5% SDS, 20 mM S-methyl methanethiosulfonate) for 20 min at 50 °C with frequent vortex. To precipitate proteins, 5 ml chilled acetone was added, and the mixture was placed at –20 °C for 30 min, followed by centrifugation at 5000 x g for 5 min. The protein pellet was then recovered, washed five times with acetone/H2O solution (70% acetone, 30% double-distilled H2O), and dissolved in 100 µl of HENS buffer (HEN buffer, 1% SDS). To reduce and label nitrothiol groups, the solution was rotated with sodium L-ascorbate (100 mM) and N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide (1.5 mM) at room temperature for 1 h, followed by the addition of chilled acetone (1 ml) to precipitate proteins. Protein pellets were washed five times with acetone/H2O solution and suspended in 100 µl of HENS buffer. To pull down biotin-labeled proteins, 200 µl of neutralization buffer (20 mM Hepes-NaOH, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, pH 7.7) and 30 µl of immobilized streptavidin beads were added into the solution, and the mixture was rotated for 1 h at room temperature. The formed streptavidin bead-protein complex was then washed five times with neutralization buffer plus NaCl (neutralization buffer with 500 mM NaCl) and two times with neutralization buffer. 30 µl of elution buffer (20 mM Hepes-NaOH, 100 mM NaCl, 1 mM EDTA, 100 mM 2-mercaptoethanol, pH 7.7) was applied to the complex to elute proteins from streptavidin beads for 20 min at room temperature. The obtained proteins were then subjected to Western blotting analysis. All of the procedures, except Western blotting, were performed in the absence of direct light. The absence of ascorbate was applied as the assay negative control.

Purification of Human Recombinant cPLA2{alpha} Protein—Affi-Gel 10-activated affinity support (catalog number 153-6099; Bio-Rad) was applied to purify recombinant human cPLA2{alpha} protein. This affinity chromatography method utilizes monoclonal anti-cPLA2{alpha} antibody, which is covalently coupled to the Affi-10 gel. In brief, the cPLA2{alpha} expression plasmid was transfected into HEK293 cells (without endogenous cPLA2{alpha} protein) for 24 h and the crude cell lysate was collected. During this period, Affi-10 gel (500 µl in 100 mM Hepes, pH 6.8) was mixed with monoclonal cPLA2{alpha} antibody (500 µg, SC-454; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2–4 h at 4 °C. The coupled gel was then washed three times with Hepes buffer (100 mM, pH 6.8) before the crude lysate was applied to the column. The lysate was passed through the column repeatedly five times, followed by three washes of the column with Hepes buffer (pH 6.8). To elute the specific protein, 100 µl of glycine-HCl (100 mM, pH 2.7) was added to the gel, and the protein was collected in a tube containing 100 µl of Hepes (100 mM, pH 8.0). The obtained protein was immediately desalted by passing through a Micro Bio-spin P-6 column (pre-equilibrated with 100 mM Hepes, pH 6.8) and then subjected to SDS-PAGE for characterization of its purity. 50 µg of purified cPLA2{alpha} protein was applied to a biotin switch assay or in vitro cPLA2 activity assay.

Site-directed Mutagenesis of the Cysteine Residues in cPLA2{alpha} Protein—The cPLA2{alpha} protein contains 9 cysteine residues (Cys-139, -141, -152, -220, -324, -331, -620, -634, and -726). These cysteine residues were mutated into alanine residues by using the QuikChange II site-directed mutagenesis kit (catalog number 200523; Stratagene, La Jolla, CA). The procedures were carried out according to the manufacturer's instructions. The primer sets for the nine mutations were as follows: Cys-139 (5'-GTC TCT TGA AGT TGC CTC ATG CCC AGA-3'; 5'-GTC TGG GCA TGA GGC AAC TTC AAG AGA C-3'); Cys-141 (5'-GAA GTT TGC TCA GCC CCA GAC CTA CG-3'; 5'-CGT AGG TCT GGG GCT GAG CAA ACT TC-3'); Cys-152 (5'-GTA TGG CTC TGG CTG ATC AGG AGA AG-3'; 5'-CTT CTC CTG ATC AGC CAG AGC CAT AC-3'); Cys-220 (5'-GGA ATT CTG GAT GCT GCT ACC TAC GTT GC-3'; GCA ACG TAG GTA GCA GCA TCC AGA ATT CC-3'); Cys-324 (5'-GTT AAT ACT GCA CAA GCC CCT TTA CCT CTT TTC; 5'-GGT GAA AAG AGG TAA AGG GGC TTG TGC AGT AT-3'); Cys-331 (5'-CCT CTT TTC ACC GCT CTT CAT GTC AAA CC-3'; 5'-GGT TTG ACA TGA AGA GCG GTG AAA AGA GG-3'); Cys-620 (5'-GCT GAA GGA GGC CTA TGT CTT TAA ACC-3'; 5'-GGT TTA AAG ACA TAG GCC TCC TTC AGC-3'); Cys-634 (5'-GGA GAA AGA TGC CCC AAC CAT CAT CC-3'; 5'-GGA TGA TGG TTG GGG CAT CTT TCT CC-3'); Cys-726 (5'-CCA TCT CGT GCC TCT GTT TCC C-3'; 5'-GGG AAA CAG AGG CAC GAG ATG G-3'). 500 ng of wild-type cPLA2{alpha} plasmid template and 125 ng of each primer were used in the PCR system for 16 cycles. Prior to transformation into XL1-Blue competent cells, the PCR product was digested for 1 h by DpnI enzyme at 37 °C to damage the wild-type template plasmid. All mutated plasmids were confirmed by DNA sequencing.

Immunoprecipitation and Western Blotting—Cells were lysed in Nonidet P-40 buffer (50 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, pH 7.2) and homogenized on ice. Cell debris was removed by centrifugation at 14,000 rpm for 20 min at 4 °C. Whole cell lysate (~500 µg in 100 µl) was recovered and incubated with mouse anti-human cPLA2{alpha} antibody (5 µl, 200 µg/ml) or mouse IgG for 1 h at 4°C, followed by the addition of protein A/G-agarose beads (30 µl) for an extra 1 h. The protein-bead complex was washed four times with Nonidet P-40 buffer and subjected to regular Western blotting. 20 µlof sample buffer was added into protein-bead complex, and the mixture was boiled at 90 °C for 5 min. All of the supernatant was loaded onto polyacrylamide gel for electrophoresis. The separated proteins in polyacrylamide gels were transferred onto nitrocellulose membrane (Bio-Rad), and the blot was blocked in 5% milk PBS-T (0.5% Tween 20 in buffer PBS) for 1 h at room temperature. The membrane was then incubated overnight with primary antibodies (1:1000) in 5% milk PBS-T. Following repeated washing with PBS-T the next day, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) for 1 h at room temperature. After washing, the blots were developed using the ECL Western blotting detection system and exposed to Eastman Kodak Co. MR radiographic films.

Statistical Analysis—Statistical analysis was performed using Microsoft Excel 2003 software. Comparisons were performed using two-tailed unpaired Student's t test. Values of p < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of iNOS Induces cPLA2{alpha} S-Nitrosylation and Activation—Given the documented role of cPLA2{alpha} in AA metabolism and the known cross-interactions between iNOS and prostaglandin signaling pathways in human cells (25, 26), we sought to determine whether iNOS could regulate AA metabolism through S-nitrosylation of cPLA2{alpha} protein. For this purpose, two human epithelial cell lines with high constitutive cPLA2{alpha} expression (SG231 and A549) were transduced with the iNOS adenovirus expression vector or the control Y5 vector to determine the occurrence of cPLA2{alpha} S-nitrosylation. As shown in Fig. 1A, forced expression of iNOS induced S-nitrosylation of cPLA2{alpha} in both SG231 cells and A549 cells. Overexpression of iNOS had no effect on cPLA2{alpha} protein level (Fig. 1A) or phosphorylation (see below). The level of cPLA2{alpha} S-nitrosylation is directly proportional to the amount of iNOS protein expressed in those cells. Furthermore, iNOS overexpression significantly increased the release of AA from both SG231 and A549 cells (Fig. 1B). These data suggest that modulation of cPLA2{alpha} by iNOS-mediated S-nitrosylation probably increases cPLA2{alpha} enzymatic activity. Indeed, in vitro cPLA2 activity assays confirmed that iNOS overexpression enhanced cPLA2 activity by 3-fold in SG231 cells and 2-fold in A549 cells (Fig. 1C). These results provide the first evidence that iNOS is able to S-nitrosylate and activate cPLA2{alpha} in human cells. The observation that the iNOS-induced increase of cPLA2 activity is blocked by the specific iNOS inhibitor, 1400W, suggests that iNOS-mediated production of NO is required for cPLA2{alpha} S-nitrosylation (Fig. 1C).

NO Donor Causes S-Nitrosylation and Activation of cPLA2{alpha}— We next utilized NO donor to evaluate the direct effect of NO on cPLA2{alpha} S-nitrosylation and enzymatic activity. The addition of the NO donor GSNO to the cell lysates isolated from SG231 cells resulted in S-nitrosylation of cPLA2{alpha}, in vitro (Fig. 2A). The assay specificity was confirmed by the observation that omission of ascorbic acid in the assay system completely abolished the detection of S-nitrosylated cPLA2{alpha}. Accordingly, incubation of cultured SG231 cells with GSNO significantly increased cPLA2 activity in vitro (Fig. 2B). Increased cPLA2 activity was also seen in the SG231 cells treated with the iNOS substrate L-Arg; this effect was blocked by the iNOS inhibitor 1400W (Fig. 2B). Furthermore, L-Arg, GSNO, and NOC-18 (another NO donor) also increased the release of AA from SG231 as well as from A549 cells (Fig. 2C). These observations document that NO donors are capable of inducing cPLA2{alpha} S-nitrosylation and activation.

We further examined the effect of NO donor in cells with a low basal level of cPLA2{alpha} protein (CCLP1). Whereas GSNO did not alter cPLA2 activity in CCLP1 cells, it increased cPLA2 activity when the CCLP1 cells were transfected with the cPLA2{alpha} expression plasmid (Fig. 2D). These findings suggest that the S-nitrosylation and activation of cPLA2{alpha} in cells is in direct proportion to the level cPLA2{alpha} protein as well as the availability of NO.

To further characterize the direct effect of NO on cPLA2{alpha} protein S-nitrosylation and to exclude the potential influence of other molecules, cPLA2{alpha} expression plasmid was transfected into HEK293 cells, and the recombinant cPLA2{alpha} protein was purified by affinity chromatography. The purified recombinant cPLA2{alpha} protein was then incubated with the NO donor to determine its S-nitrosylation and enzyme activity, in vitro. As shown in Fig. 3A, GSNO induces the S-nitrosylation of purified cPLA2{alpha} protein. Accordingly, the NO donors (GSNO and NOC-18) also increased the enzyme activity of purified cPLA2{alpha} (Fig. 3B). These results demonstrate that NO is capable of directly S-nitrosylating and activating cPLA2{alpha}.

The iNOS-induced cPLA2{alpha} S-Nitrosylation and AA Release Is Enhanced by COX-2—In light of the documented functional coupling between cPLA2{alpha} and COX-2 in human cells (5, 21–24), we further examined whether COX-2 might influence the iNOS-induced cPLA2{alpha} S-nitrosylation. For this purpose, HEK293 cells were transfected with the expression vector for cPLA2{alpha}, COX-2 and iNOS, either alone or in combination, to determine the S-nitrosylation of cPLA2{alpha}. HEK293 cells were utilized because they do not constitutively express cPLA2{alpha}, COX-2, or iNOS and allow high transfection efficiency. As shown in Fig. 4A, although S-nitrosylation of cPLA2{alpha} was observed in HEK293 cells cotransfected with the iNOS and cPLA2{alpha} vectors, much more S-nitrosylated cPLA2{alpha} is detected with the combination of COX-2 transfection. These findings indicate that COX-2 has the ability to enhance iNOS-induced cPLA2{alpha} S-nitrosylation. To further examine the effect of endogenous COX-2 on cPLA2{alpha} S-nitrosylation, HEK293 cells were treated with the cytokine mixture containing IL-1β, TNF-{alpha}, and IFN-{gamma} to induce COX-2 expression (these cytokines are known to induce COX-2 gene expression in other cells). As expected, treatment of the HEK293 cells with the cytokine mixture effectively induced the expression of COX-2, without alteration of cPLA2{alpha} expression, cPLA2{alpha} phosphorylation, or iNOS expression (Fig. 4B). Notably, the combination of iNOS expression with induction of endogenous COX-2 led to dramatic increase of cPLA2{alpha} S-nitrosylation (Fig. 4C). This effect was blocked by the iNOS inhibitor, 1400W (Fig. 4C), further suggesting the requirement of iNOS activity for cPLA2{alpha} S-nitrosylation. Accordingly, the release of AA was also dramatically enhanced by the combination of iNOS overexpression and COX-2 induction (~20-fold), compared with a 4-fold increase in the absence of COX-2 (Fig. 4D). Collectively, these findings demonstrate that COX-2 facilitates iNOS-induced cPLA2{alpha} S-nitrosylation.


Figure 1
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  		FIGURE 1.
Effect of iNOS overexpression on cPLA2{alpha} S-nitrosylation and activity. A, iNOS overexpression induces S-nitrosylation of cPLA2{alpha}. SG231 and A549 cells were infected with different titers of iNOS or control Y5 adenovirus for 24 h, and cell lysates were collected to determine cPLA2{alpha} S-nitrosylation by biotin switch as described under "Experimental Procedures." The same samples were also subjected to regular Western blotting for detection of total cPLA2{alpha} and iNOS. β-Actin was utilized to normalize protein loading. B, iNOS overexpression increases AA release. SG231 and A549 cells infected with different titers of Y5 or iNOS adenovirus were incubated with [5,6,8,9,11,12,14,15-3H]arachidonic acid (0.5 µCi/ml culture medium) for 24 h. The cells were then washed five times to measure AA release as described under "Experimental Procedures" (*, p < 0.05 compared with the same titer Y5). C, iNOS overexpression enhances cPLA2 activity in vitro. SG231 and A549 cells were infected with Y5 (100 MOI for SG231 cells, 500 MOI for A549 cells) or iNOS adenovirus for 24 h with or without 1400W (100 µM) and then subjected to a cPLA2 activity assay in vitro (a, p < 0.05 compared with Y5; b, p < 0.01 compared with lower titer iNOS or Y5; c, p < 0.01 compared with higher titer iNOS alone; n = 3).

 
The Activation of Endogenous cPLA2{alpha} Is Enhanced by Combination of iNOS Expression and COX-2 Induction—After the effect of iNOS and COX-2 on the S-nitrosylation and activation of transfected (exogenous) cPLA2{alpha} was documented in the HEK393 cells, we further evaluated the effect of combined iNOS and COX-2 on the activation of endogenously expressed cPLA2{alpha} in SG231 cells (with a high level of constitutive cPLA2{alpha} expression). As shown in Fig. 5A, cytokine treatment of SG231 cells also induced the expression of COX-2 protein, with no effect on the level of cPLA2{alpha} protein, cPLA2{alpha} phosphorylation, or iNOS expression. Combination of iNOS expression and COX-2 induction resulted in an ~24-fold increase of AA release in SG231 cells, which was in contrast to the 3-fold increase with iNOS expression alone (Fig. 5B). These results further reveal the synergistic effect of COX-2 and iNOS on cPLA2{alpha} activation.

Physical Association between iNOS and cPLA2{alpha}: Effect of COX-2—NO is a highly diffusible and short lived physically active gas. The proximity between NOS and targeted proteins has been shown to help form protein S-nitrosylation (27, 36). We utilized immunoprecipitation and Western blot analysis to determine the association between cPLA2{alpha} and iNOS. In HEK293 cells, the cPLA2{alpha}-iNOS association complex was detected in cells with forced expression of both cPLA2{alpha} and iNOS but not in cells with expression of either molecule alone (Fig. 6A). In SG231 cells, the cPLA2{alpha}-iNOS association complex was detected in cells transduced with the iNOS adenovirus vector but not in cells infected with the Y5 control vector (Fig. 6B). These findings indicate the proximity between cPLA2{alpha} and iNOS in cells, which may explain the apparent effect of iNOS on cPLA2{alpha} S-nitrosylation. Importantly, COX-2 induction increased the amount of cPLA2{alpha} immunoprecipitated by iNOS in both HEK293 and SG231 cells (Fig. 6, A and B). Therefore, COX-2 enhances the association between iNOS and cPLA2{alpha}; this effect may explain the synergistic effect of COX-2 on iNOS-induced cPLA2{alpha} S-nitrosylation. Since the COX-2 inhibitor, NS398, failed to prevent COX-2-induced association between iNOS and cPLA2{alpha} (Fig. 6C), it is likely that COX-2 may facilitate iNOS-cPLA2{alpha} association through COX-2 protein itself rather than through its enzymatic products.


Figure 2
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  		FIGURE 2.
The effect of exogenous NO on cPLA2{alpha} S-nitrosylation, activity, and AA release. A, the NO donor, GSNO, induces cPLA2{alpha} S-nitrosylation, in vitro. The cell lysates isolated from SG231 cells were incubated with GSNO (20 µM) for 30 min. The samples were then subjected to the biotin switch assay in the presence or absence of ascorbate to determine cPLA2{alpha} S-nitrosylation. B, the effect of NO donor and iNOS substrate on cPLA2 activity. SG231 cells were incubated with GSNO (20 µM), L-arginine (1 mM) with or without 1400W (100 µM) for 30 min. The cell lysate was then collected and subjected to a cPLA2 activity assay (a, p < 0.05 compared with vehicle; b, p < 0.05 compared with L-arginine treatment; n = 3). C, the effect of NO donor and iNOS substrate on AA release. SG231 and A549 cells were incubated with [5,6,8,9,11,12,14,15-3H]arachidonic acid (0.5 µCi/ml culture medium) for 24 h. The cells were then washed five times with culture medium, followed by incubation with GSNO (20 µM), NOC-18 (100 µM), L-Arg (1 mM), or vehicle Me2SO for 1 h. The supernatant was collected to measure 3H radioactivity (*, p < 0.05 compared with vehicle; n = 3). D, the effect of NO donor on cPLA2 activity in CCLP1 cells. CCLP1 cells were transfected with the cPLA2{alpha} expression plasmid or the control MT2 plasmid for overnight. The cell lysate was then collected and incubated with GSNO (20 µM) or N Gmonomethyl-L-arginine (250 µM) for 30 min at 4 °C. The samples were subsequently analyzed for cPLA2 activity as described under "Experimental Procedures" (a, p < 0.05 compared with MT2; b, p < 0.05 compared with cPLA2{alpha} transfection plus vehicle Me2SO treatment; n = 3).

 


Figure 3
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  		FIGURE 3.
NO donor induces the S-nitrosylation and activation of purified cPLA2{alpha}. A, the NO donor, GSNO, induces S-nitrosylation of purified cPLA2{alpha}, in vitro. HEK293 cells were transfected with the cPLA2{alpha} expression plasmid for 24 h. Prior to the biotin switch assay, cPLA2{alpha} protein was purified from the crude cell lysates and exposed to GSNO (20 µM) for 30 min. B, the NO donors, GSNO and NOC-18, enhance the activity of purified cPLA2{alpha}, in vitro. HEK293 cells were transfected with the cPLA2{alpha} expression plasmid, and the cPLA2{alpha} protein was purified from the crude lysates as described under "Experimental Procedures." The purified cPLA2{alpha} protein was then subjected to a cPLA2 activity assay in vitro (*, p < 0.01 versus vehicle; n = 3).

 
In light of the reported binding of COX-2 to iNOS (27), we performed further immunoprecipitation and Western blot analysis to examine the potential association between cPLA2{alpha} and COX-2 in our system. As shown in Fig. 6D, cPLA2{alpha} is associated with COX-2 in SG231 cells transduced with the iNOS adenoviral expression vector plus cytokine treatment; this association is minimal in the absence of iNOS. These observations suggest an important role of iNOS for the association between cPLA2{alpha} and COX-2. Therefore, it is possible that iNOS may recruit cPLA2{alpha} and COX-2 to form a multiprotein complex.


Figure 4
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  		FIGURE 4.
The effect of iNOS and COX-2 on the S-nitrosylation and activation of cPLA2{alpha} in HEK293 cells. A, overexpression of COX-2 enhances iNOS-induced cPLA2{alpha} S-nitrosylation. HEK293 cells were transfected 24 h with the cPLA2{alpha} expression plasmid, COX-2 expression plasmid, or the control MT2 plasmid, with or without iNOS adenovirus infection (50 MOI). The cell lysates were subjected to a biotin switch assay to determine cPLA2{alpha} S-nitrosylation and conventional Western blotting to determine the input of cPLA2{alpha}, COX-2, and iNOS. B, induction of COX-2 by cytokine mixture. HEK293 cells with or without overexpression of cPLA2{alpha} or iNOS were treated with the cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cell lysates were collected for SDS-PAGE and Western blotting. C, induction of COX-2 enhances iNOS-induced cPLA2{alpha} S-nitrosylation. HEK293 cells transfected with cPLA2{alpha} expression plasmid or the control MT2 plasmid or infected with iNOS or Y5 adenovirus were treated with cytokine mixture for 24 h. Some cells were treated with the iNOS inhibitor, 1400W (100 µM). The cells were processed for biotin switch assay and conventional Western blotting. D, effect of iNOS, cPLA2{alpha}, and COX-2 on AA release. HEK293 cells labeled with [3H]AA were transfected with cPLA2{alpha} expression plasmid or the control MT2 plasmid, infected with iNOS or Y5 adenovirus, or treated with the cytokine mixture for 24 h. At the end of treatment, the cells were washed five times, followed by culture with regular medium containing 0.5% FBS for 1 h. The culture medium was then collected for [3H]AA measurement using a liquid scintillation counter (a, p < 0.05 compared with MT2 transfection; b, p < 0.01 compared with cPLA2{alpha} + Y5 transfection; c, p < 0.01 compared with cPLA2{alpha} + Y5 + cytokines; d, p < 0.01 compared with cPLA2{alpha} + iNOS + cytokines; n = 3).

 
iNOS Induces Maximal PGE2 Production in the Presence of cPLA2{alpha} and COX-2—Since cPLA2{alpha} activation leads to the release of AA, providing substrate for COX-2-mediated PGE2 synthesis, we further examine the effect of iNOS, cPLA2{alpha}, and COX-2 in different combinations on PGE2 production. In HEK293 cells, combined iNOS expression and COX-2 induction in the absence of cPLA2{alpha} induced only less than a 2-fold increase of PGE2 (Fig. 7). When the cells were transfected with the cPLA2{alpha} expression vector, either iNOS expression or COX-2 induction induced a 2–3-fold increase of PGE2. Interestingly, the combination of cPLA2{alpha}, COX-2, and iNOS dramatically enhanced PGE2 production by more than 80-fold (Fig. 7). It is noteworthy that overexpression of cPLA2{alpha} and iNOS in combination with COX-2 induction achieved a much higher increase of PGE2 production than either iNOS plus cPLA2{alpha} or cPLA2{alpha} plus COX-2. A similar synergistic effect was also observed in the cells with endogenous expression of cPLA2{alpha} (SG231 and A549) (Fig. 8). iNOS overexpression plus COX-2 induction increased the production of PGE2 by nearly 40-fold in SG231 cells (Fig. 8A) and ~100-fold in A549 cells (Fig. 8B). These results demonstrate that maximal PG synthesis is achieved by the synergistic interactions among iNOS, cPLA2{alpha}, and COX-2.

Cys-152 of cPLA2{alpha} Is Essential for S-Nitrosylation—Upon completion of the above experiments, we further characterized the contribution of individual cysteine residues of cPLA2{alpha} to iNOS-induced S-nitrosylation. To this end, site-directed mutagenesis was performed to replace each of the 9 cysteine residues with alanine (Cys-139, -141, -152, -220, -324, -331, -620, -634, and -726). The generated Cys-mutated cPLA2{alpha} expression plasmid or wild type vector was transfected into HEK293 cells to determine cPLA2{alpha} S-nitrosylation, enzyme activity, and AA release. As shown in Fig. 9A, mutation of Cys-152 markedly reduced the iNOS-induced cPLA2{alpha} S-nitrosylation, whereas mutation of other cysteine residues exhibited no apparent effect. Accordingly, mutation of Cys-152, but not other cysteine residues, prevented iNOS-induced cPLA2{alpha} activation (Fig. 9B) and arachidonic acid release (Fig. 9C). These results demonstrate that Cys-152 is the primary site for iNOS-induced cPLA2{alpha} S-nitrosylation and enzymatic activation.


Figure 5
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  		FIGURE 5.
Influence of iNOS and COX-2 on the activation of endogenous cPLA2{alpha} in SG231 cells. A, induction of COX-2 by cytokines. SG231 cells were infected with Y5 (100 MOI) or iNOS (100 MOI) adenovirus for 24 h, with or without cytokine mixture treatment (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}). The cell lysates were subjected to SDS-PAGE and Western blotting to determine the levels of cPLA2{alpha}, cPLA2{alpha} phosphorylation, COX-2, iNOS, and β-actin. B, COX-2 induction enhances iNOS-induced AA release. SG231 cells were subjected to the same treatment as indicated in A. The release of AA was measured according to the method described under "Experimental Procedures" (a, p < 0.01 versus Y5; b, p < 0.05 versus vehicle Me2SO; c, p < 0.01 versus cytokines + Y5 or iNOS + vehicle; n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
NO is a bioactive molecule with a single unpaired electron. The two major mechanisms whereby NO influences its intracellular targets are stimulation of guanylyl cyclase by direct binding of NO to iron in heme at the active site of guanylyl cyclase (37) and S-nitrosylation of protein targets on appropriate cysteines (34, 36, 38). S-Nitrosylation refers to the NO adduct with cysteine residue; it is increasingly recognized as an important posttranslational modification mechanism that mediates the diverse actions of NO. Our data in this study demonstrate that iNOS/NO S-nitrosylates and activates cPLA2{alpha}, which represents a novel mechanism for the regulation of cPLA2{alpha} in human cells. This assertion is based on the following observations: 1) iNOS overexpression induces cPLA2{alpha} S-nitrosylation, enhances its catalytic activity, and increases the release of AA from cells; 2) mutation of Cys-152 of cPLA2{alpha} abolishes iNOS-induced cPLA2{alpha} S-nitrosylation and enzymatic activation; 3) the level of cPLA2{alpha} S-nitrosylation is in direct proportion to the amount of iNOS and cPLA2{alpha} proteins expressed in the cells; 4) the iNOS-induced increase of cPLA2 activity is blocked by the specific iNOS inhibitor, 1400W; 5) the addition of the NO donor, GSNO, to isolated cell lysates induces S-nitrosylation of cPLA2{alpha}, in vitro; 6) incubation of cultured cells with GSNO significantly increases cPLA2 activity; 7) exposure of purified cPLA2{alpha} protein to NO donor causes its S-nitrosylation and activation; 8) the iNOS substrate L-arginine increases cPLA2 activity, and the effect is blocked by the iNOS inhibitor; 9) L-arginine and NO donors also increase the release of AA from cells; and 10) cPLA2{alpha} is physically associated with iNOS.


Figure 6
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  		FIGURE 6.
cPLA2{alpha}, iNOS, and COX-2 are physically associated. A, co-immunoprecipitation (IP) of cPLA2{alpha} with iNOS in HEK293 cells. HEK293 cells were transfected with cPLA2{alpha} or MT2 plasmid, infected with iNOS (50 MOI) or Y5 adenovirus, or treated with cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cell lysates were collected and incubated with anti-cPLA2{alpha} antibody or mouse IgG (negative control) for 1 h at 4 °C, followed by incubation with protein A/G-agarose beads for 40 min. After five washes with lysis buffer, the protein-bound beads were subjected to Western blotting (WB) for detection of iNOS. B, co-immunoprecipitation of cPLA2{alpha} with iNOS in SG231 cells. SG231 cells were transfected with iNOS (100 MOI) or control Y5 adenovirus and/or treated with cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) or vehicle Me2SO for 24 h. The cell lysates were then processed for immunoprecipitation and Western blot analysis. C, the effect of the COX-2 inhibitor, NS-398, on cPLA2{alpha} and iNOS association in SG231 cells. SG231 cells were infected with iNOS adenovirus (100 MOI), with or without cytokines (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) or NS-398 (25 µM) treatment for 24 h. The cell lysates were collected and subjected to immunoprecipitation and Western blotting. D, effect of iNOS on cPLA2{alpha} and COX-2 association. SG231 cells were exposed to cytokines (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) with or without infection of iNOS adenovirus (100 MOI) for 24 h. The cell lysates were then collected and subjected to cPLA2{alpha} immunoprecipitation and COX-2 Western blotting. Each figure is representative of three separated experiments.

 


Figure 7
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  		FIGURE 7.
Maximal PGE2 production by a combination of iNOS, cPLA2{alpha}, and COX-2 in HEK293 cells. HEK293 cells labeled with [3H]AA were infected with iNOS (50 MOI) or Y5 (50 MOI) adenovirus or transfected with cPLA2{alpha} or MT2 plasmid in the presence or absence of cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cell culture supernatant was then collected to measure PGE2 production (a, p < 0.01 compared with Y5 + cPLA2{alpha}; b, p < 0.01 compared with iNOS + cPLA2{alpha} + vehicle or iNOS + cytokines + MT2; c, p < 0.01 compared with Y5 + cPLA2{alpha} + vehicle; n = 3). This figure is representative of three experiments.

 
The observation that NO donor induces cPLA2{alpha} S-nitrosylation in isolated cell lysates and purified recombinant protein in vitro suggests direct modification of cPLA2{alpha} by NO adduct. This is also supported by the results from cultured cells showing that overexpression of iNOS or the addition of iNOS substrate or NO donor induced cPLA2{alpha} S-nitrosylation and activation and that specific iNOS inhibitor blocked the iNOS overexpression or substrate-induced cPLA2{alpha} activation. Our data show that S-nitrosylated cPLA2{alpha} exhibits enhanced enzymatic activity. This observation is consistent with other studies showing that S-nitrosylated proteins display alterations in enzymatic activity, trafficking through intracellular compartments, stability in intracellular environment, binding with proteins, and other functions (34, 36, 39–45).

The occurrence of S-nitrosylation and concomitant modulation of protein function are regulated by factors including NO concentration fluctuation, other redox signaling, proximity of NO with targeted protein, intracellular location of protein, iron concentration, and the presence of denitrosylation, thus far beyond control and prediction under random conditions (46). In this study, the association between iNOS and cPLA2{alpha} proteins provides a mechanistic explanation for the noticeable effect of iNOS on cPLA2{alpha} S-nitrosylation, which is consistent with the documented proximity between NOS and other targeted proteins for efficient S-nitrosylation (27, 36). We observed that transduction of iNOS adenovirus vector led to more S-nitrosylation of cPLA2{alpha} than NO donors; this effect is most likely due to the higher concentration of NO accumulated at specific organelle or intracellular membrane in iNOS-overexpressed cells or due to the physical interaction between iNOS and cPLA2{alpha}, which allows efficient transfer of produced NO to cPLA2{alpha}.


Figure 8
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  		FIGURE 8.
Maximal PGE2 production by combination of iNOS and COX-2 in cells with endogenous expression of cPLA2{alpha}. A, the effect of iNOS and COX-2 on PGE2 production in SG231 cells. SG231 cells labeled with [3H]AA were infected with iNOS (100 MOI) or Y5 adenovirus and treated with cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cell culture supernatant was collected and subjected to a PGE2 assay (a, p < 0.01 compared with Y5 + cytokines or iNOS + vehicle; b, p < 0.05 compared with Y5 + vehicle; n = 3). B, effect of iNOS and COX-2 on PGE2 production in A549 cells. A549 cells labeled with [3H]AA were infected with iNOS (500 MOI) or Y5 (500 MOI) adenovirus with or without cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The culture supernatant was collected for the PGE2 assay (a, p < 0.01 compared with Y5; b, p < 0.01 compared with Y5 + cytokines; c, p < 0.01 compared with Y5 + vehicle, n = 3).

 


Figure 9
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  		FIGURE 9.
Cys-152 of cPLA2{alpha} is critical for S-nitrosylation. Site-directed mutagenesis was performed to replace each of the nine cysteine residues of cPLA2{alpha} (Cys-139, -141, -152, -220, -324, -331, -620, -634, and -726). The generated mutants and wild type cPLA2{alpha} expression plasmid were then transfected into HEK293 cells to determine cPLA2{alpha} S-nitrosylation, enzyme activity, and the release of arachidonic acid. A, Cys-152 mutation prevents iNOS-induced cPLA2{alpha} S-nitrosylation. HEK293 cells were transfected with the mutated or wild-type cPLA2{alpha} expression plasmid and infected with iNOS adenovirus (100 MOI) for 24 h. The cell lysates were collected and subjected to a biotin switch assay to detect cPLA2{alpha} S-nitrosylation. Regular Western blotting was performed to determine the levels of cPLA2{alpha} and iNOS proteins. B, mutation of Cys-152 prevents iNOS-induced of cPLA2{alpha} activity. HEK293 cells transfected with the mutated or wild-type cPLA2{alpha} expression plasmid were infected with iNOS adenovirus (100 MOI) and exposed to cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cell lysates were subjected to a cPLA2{alpha} activity assay, in vitro, as described under "Experimental Procedures" (*, p < 0.01 versus wild-type cPLA2{alpha}; n = 3). C, mutation of Cys-152 prevents iNOS-induced AA release. HEK293 cells transfected with the mutated or wild-type cPLA2{alpha} expression plasmid were infected with iNOS adenovirus (100 MOI) and exposed to cytokine mixture (50 ng/ml IL-1β, 50 ng/ml TNF-{alpha}, 100 ng/ml IFN-{gamma}) for 24 h. The cells were analyzed for the release of [3H]arachidonic acid as described under "Experimental Procedures" (*, p < 0.01 versus wild-type cPLA2{alpha}; n = 3).

 
Another important aspect of this study is the dramatic synergistic effect of COX-2 on iNOS-induced cPLA2{alpha} S-nitrosylation and activation. Our results show that although cPLA2{alpha} S-nitrosylation is present in cells expressing iNOS and cPLA2{alpha}, the level of S-nitrosylated cPLA2{alpha} is much higher in the presence of COX-2. For example, in HEK283 cells, a combination of iNOS, cPLA2{alpha}, and COX-2 induced a 20-fold increase of AA release, in contrast to the 4-fold increase induced by iNOS plus cPLA2{alpha}. Accordingly, the combination of cPLA2{alpha}, iNOS, and COX-2 dramatically enhanced PGE2 production by more than 80-fold, in contrast to the 3-fold increase by iNOS plus cPLA2{alpha} and less than 2-fold increase by COX-2 plus iNOS. A similar synergistic effect was also seen in two other epithelial cells (SG231 and A549). These findings indicate that COX-2 has the ability to enhance iNOS-induced cPLA2{alpha} S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interactions among iNOS, cPLA2{alpha}, and COX-2. It is worth mentioning that COX-2-mediated cPLA2{alpha} activation requires the presence of iNOS, further highlighting the synergistic effect of COX-2 on iNOS-induced cPLA2{alpha} S-nitrosylation.

Since COX-2 increases the formation of cPLA2{alpha}-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA2{alpha} S-nitrosylation may be mediated at least in part through increased association between iNOS and cPLA2{alpha}. However, it is not clear at this time whether cPLA2{alpha} directly binds to COX-2. We were able to coimmunoprecipitate COX-2 with cPLA2{alpha} antibody in cells expressing cPLA2{alpha}, COX-2, and iNOS but not in cells without iNOS. Thus, it is possible that a multiprotein complex is formed, containing these three proteins with or without other molecules. Given the documented direct binding between COX-2 and iNOS (27) and our observation that iNOS is essential for coimmunoprecipitation of cPLA2{alpha} and COX-2, it is likely that iNOS may be the central molecule that recruits cPLA2{alpha} and COX-2 to the binding complex.

The synergistic effect among iNOS, cPLA2{alpha}, and COX-2 for PGE2 synthesis is highly significant, given that cPLA2{alpha} and COX-2 are rate-limiting key enzymes for PG synthesis and that iNOS is the key enzyme for production of NO. Under inflammatory processes, activation of these key enzymes results in simultaneous production of NO and PGs. Therefore, it is possible that the illustrated interaction among iNOS, cPLA2{alpha}, and COX-2 may importantly contribute to the production of large amounts of PGs and NO that simultaneously drive and perpetuate inflammatory processes.

In summary, our results presented in this study demonstrate a novel iNOS/NO-mediated cPLA2{alpha} S-nitrosylation and activation for prostaglandin synthesis and the striking synergistic effect of COX-2 in this process. Given the high magnitude of PGE2 production generated by the synergistic interaction among iNOS, cPLA2{alpha}, and COX-2, it is possible that this mechanism may be implicated in the synthesis of bioactive prostaglandins during disease processes, such as inflammation and carcinogenesis. It is conceivable that disruption of the association among cPLA2{alpha}, iNOS, and COX-2 may achieve more effective inhibition of AA release and PGE2 synthesis than conventional pharmacological inhibitors. This intervention may represent a highly effective therapeutic approach that merits further investigation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health R01 Grants CA102325 and CA106280 (to T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pathology, University of Pittsburgh School of Medicine, MUH E-740, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-647-9504; Fax: 412-647-5237; E-mail: wut{at}upmc.edu.

2 The abbreviations used are: cPLA2{alpha}, cytosolic phospholipase A2{alpha}; AA, arachidonic acid; COX, cyclooxygenase; GSNO, S-nitrosoglutathione; IFN-{gamma}, interferon-{gamma}; IL-1β, interleukin-1β; iNOS, inducible nitric-oxide synthase; NO, nitric oxide; PG, prostaglandin; PGE2, prostaglandin E2; PLA2, phospholipase A2; TNF-{alpha}, tumor necrosis factor-{alpha}; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; MOI, multiplicity of infection; FBS, fetal bovine serum. Back


   ACKNOWLEDGMENTS
 
We thank Drs. James Clark and Timothy Hla for providing the human cPLA2{alpha} and COX-2 expression plasmids and the University of Pittsburgh Vector Core Facility for the iNOS adenovirus vector.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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