IPLA2B REGULATES VIRUS-INDUCED INDUCIBLE NO SYNTHASE EXPRESSION BY MACROPHAGES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。最近的证据支持钙非依赖性磷脂酶A2 （iPLA2）在巨噬细胞诱导一氧化氮合酶（iNOS）表达的抗病毒反应中的调节作用。由于iPLA2的两个哺乳动物亚型（iPLA2B和iPLA2?）已经被克隆和鉴定，本研究的目的是鉴定巨噬细胞中在病毒感染反应中调节iNOS表达的特异性亚型。溴烯醇内酯（BEL）是iPLA2的自杀底物抑制剂，在低微摩尔浓度下抑制这两种异构体的活性。然而，BEL的R-和s -对映体对iPLA2？和iPLA2B。在这项研究中，我们发现iPLA2B-selective (S)-BEL在巨噬细胞中以浓度相关的方式抑制脑心肌炎病毒（EMCV）诱导的iNOS表达、一氧化氮产生和iPLA2酶活性，其抑制特性与外消旋bel的抑制特性非常相似。cAMP反应元件结合蛋白（CREB）是iPLA2的一个下游靶点，是响应病毒感染时iNOS转录激活所必需的。与BEL对映体对iNOS表达的影响一致，巨噬细胞中(S)-BEL比(R)-BEL更有效地抑制emcv诱导的CREB磷酸化。利用从iPLA2B缺失小鼠中分离的巨噬细胞，病毒感染不能刺激iNOS mRNA的积累和蛋白的表达，从而为emcv诱导的iNOS表达需要iPLA2B提供了遗传学证据。这些发现为iPLA2B在病毒诱导的巨噬细胞iNOS表达中的信号作用提供了证据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 (iPLA2B and iPLA2?) 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 iPLA2? and iPLA2B, respectively. In this study, we show that the iPLA2B-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 iPLA2B-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2B is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2B in virus-induced iNOS expression by macrophages.