Genetic and pharmacologic evidence that calcium-independent phospholipase A₂β regulates virus-induced inducible nitric-oxide synthase expression by macrophages

Recent evidence supports a regulatory role for the calcium-independent phospholipase A₂ (iPLA₂) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA₂ (iPLA₂β and iPLA ₂γ) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA₂, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display ∼10-fold greater potency for inhibition of the enzymatic activity of iPLA₂γ and iPLA₂β, respectively. In this study, we show that the iPLA₂β-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA₂ enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA₂ that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA₂β-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA₂β is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA₂β in virus-induced iNOS expression by macrophages.