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J. Biol. Chem., Vol. 283, Issue 6, 3077-3087, February 8, 2008

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Activation of Cytosolic Phospholipase A₂ through Nitric Oxide-induced S-Nitrosylation

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

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

Cytosolic phospholipase A₂ (cPLA₂) 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 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 S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA₂ 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 protein induced S-nitrosylation of cPLA₂ in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA₂ activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA₂ in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA₂ is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA₂ 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 S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA₂, and COX-2. Since COX-2 enhances the formation of cPLA₂-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA₂ S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA₂. These findings disclose a novel link among cPLA₂, iNOS, and COX-2, which form a multiprotein complex leading to cPLA₂ 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.

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

^* 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.

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.

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