Activation of cytosolic phospholipase A2alpha through nitric oxide-induced S-nitrosylation. Involvement of inducible nitric-oxide synthase and cyclooxygenase-2.
Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)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 cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)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 cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) 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 cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.
Xu
Lihong
L
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
Han
Chang
C
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
Lim
Kyu
K
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
Wu
Tong
T
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213. Electronic address: wut@upmc.edu.
D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D054513:Group IV Phospholipases A2 / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D014162:Transfection
D016718:Arachidonic Acid; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D054513:Group IV Phospholipases A2; D009584:Nitrogen