IPLA2B REGULATES VIRUS-INDUCED INDUCIBLE NO SYNTHASE EXPRESSION BY MACROPHAGES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。最近的证据支持钙非依赖性磷脂酶A2(IPLA2)在巨噬细胞诱导型一氧化氮合酶(INOS)表达的抗病毒反应中的调节作用。因为iPLA2的两种哺乳动物亚型(iPLA2B和iPLA2？)本研究的目的是鉴定巨噬细胞中调节诱导型一氧化氮合酶表达的特异性异构体(S)。IPLA2的自杀性底物抑制剂溴烯醇内酯(BEL)在低微摩尔浓度下抑制这两种异构体的活性。而BEL的R-和S-对映体对iPLA2？和iPLA2B。在本研究中，我们发现选择性的iPLA2B(S)-BEL抑制脑心肌炎病毒诱导的巨噬细胞诱导型一氧化氮合酶的表达、一氧化氮的产生和iPLA2的酶活性，这种抑制作用与消旋的BEL的抑制特性非常相似。CAMP反应元件结合蛋白(cAMP Response Element-BindingProtein，CREB)是iPLA2下游靶点之一，与BEL对诱导型一氧化氮合酶表达的影响一致，(S)-BEL-BEL比(R)-BEL更有效地抑制EMCV诱导的CREB磷酸化。使用从iPLA2B缺失小鼠分离的巨噬细胞，病毒感染未能刺激iNOS mRNA积累和蛋白表达，从而提供了遗传证据，表明iPLA2B是EMCV诱导iNOS表达所必需的。这些发现为iPLA2B在病毒诱导的巨噬细胞iNOS表达中发挥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.