Study of brain arachidonic acid metabolism using knockout mice

使用基因敲除小鼠进行脑花生四烯酸代谢研究

基本信息

批准号：
7592001
负责人：
francesca bosetti
金额：
$ 10.15万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7592001
关键词：
ATP Citrate (pro-S)-Lyase Ablation Acetyl-CoA C-Acetyltransferase Affect Anti Inflammatory Analgesics Arachidonic Acids Brain Brain region Cerebral cortex Coenzyme A Complement Factor B Cytokine Signaling Data Dinoprostone Down-Regulation Exhibits GABA transporter GABA-A Receptor GAT3 transporter GDP dissociation inhibitor Gene Expression Gene Expression Microarray Analysis Gene Expression Profile Gene Expression Regulation Genes Genetic Genetic Transcription Genotype Goals Guanine Nucleotide Dissociation Inhibitors Hand Hippocampus (Brain)Homocysteine Homocystine Hydrolase Individual JAK1 gene JAK2 gene Janus kinase Janus kinase 2 Knockout Mice Membrane Proteins Messenger RNA Metabolic Metabolism Methionine Methionine Metabolism Pathway Microarray Analysis Mitochondria Molecular Mus NF-kappa B Non-Steroidal Anti-Inflammatory Agents Nuclear Oxidoreductase Play Polymerase Chain Reaction Prostaglandin-Endoperoxide Synthase Prostaglandins Protein Isoforms Proteins Regulation Ring Finger Domain Role Signal Pathway Sterol O-Acyltransferase Therapeutic Thinking Time Up-Regulation Validation Wild Type Mouse Work cohort cyclooxygenase 1 cyclooxygenase 2 gamma-Aminobutyric Acid high throughput technology lipid metabolism methionine adenosyltransferase neurotransmission oxidation rho transcription factor

项目摘要

COX-1 and COX-2 produce prostanoids from arachidonic acid (AA) and are thought to have important yet distinct roles in normal brain function. Deletion of COX-1 or COX-2 results in profound differences both in brain levels of prostaglandin E2 (PGE2)and in activation of the transcription factor nuclear factor-B (NF-kB), suggesting that COX-1 and COX-2 play distinct roles in brain arachidonic acid metabolism and regulation of gene expression. To further elucidate the role of COX isoforms in the regulation of the brain transcriptome, we performed microarray analysis of gene expression in the cerebral cortex and hippocampus of mice deficient in COX-1 (COX-1 KO) or COX-2 (COX-2 KO). Although a majority of the genes (>93%) shown to be differentially expressed in the separate brain regions of COX-1 KO or COX-2 KO mice only occurred in mice null for one isoform and not the other, the expression of some genes was changed in both COX-1 KO and COX-2 KO mice. In the cerebral cortex, there were four genes with altered expression in both COX-1 KO and COX-2 KO mice. Of these four, Rho-GDP dissociation inhibitor exhibited increased expression in both genotypes. The expression of the other three genes (mitochondrial inner membrane protein, GABA transporter GAT3, and ring finger protein 24) changed in opposite directions (downregulated in COX-1 KO and upregulated in COX-2 KO mice), suggesting an isoform-specific effect on expression of these genes. Such an isoform-specific effect was not observed in the hippocampus, where all 12 genes whose expression was altered in both COX-1 KO and COX-2 KO mice exhibited changes in a similar direction in both genotypes when compared with wild-type mice, suggesting that this cohort of genes is responsive to a general alteration in AA metabolism that is not specific to COX-1 or COX-2. In cerebral cortex of COX-2 KO mice, we identified increased expression of three genes that work in tandem in the final steps of short chain lipid metabolism by oxidation, namely hydroxyacyl-coenzyme A dehydrogenase type II (HADH2), acetyl-coenzyme A acetyltransferase 1 (ACAT1) and ATP citrate lyase (ACLY). Gene expression of ACAT 1, but not that of HADH2 and ACLY, was found to be decreased in the hippocampus of COX-1 KO mice. Expression of methionine adenosyltransferase II (MAT2B) and adenosylhomocysteine hydrolase (AHCY), two genes that work in tandem to metabolize methionine to homocysteine, was increased in cerebral cortex of COX-2 KO mice. Expression of MAT2B, but not of AHCY, was decreased in COX-1 KO hippocampus. Expressions of two genes involved in GABA neurotransmission were altered in cerebral cortex and hippocampus of COX KO mice. Microarray analysis of GABA-A receptor subunit 1 (GABRB1) in the hippocampus of COX-2 KO mice demonstrated downregulation of gene expression. Validation with Q-PCR demonstrated that the expression of GABRB1 was not only decreased in hippocampus of COX-2 KO mice but it was also decreased in cerebral cortex of COX-2 KO mice. GABA transporter (GAT)3 expression in cerebral cortex of COX KO mice exhibited a genotype-specific effect. COX-1 KO mice demonstrated downregulation of gene expression whereas COX-2 KO showed upregulation of mRNA level. Janus kinase (JAK) isoforms 1 and 2 were found to be expressed in a genotype-dependent manner in hippocampus. COX-1 KO mice had increased expression of JAK1 but not of JAK2. On the other hand, JAK-2 expression was decreased in COX-2 KO but not in COX-1 KO mice. Overall, our findings suggest that ablation of COX activity alters the transcription of many genes, including those involved in oxidation, methionine metabolism, GABA neurotransmission, and cytokine signaling. Although some of the molecular mechanisms underlying these changes are not well understood at this time, these data identify metabolic and signaling pathways that were previously not known to be affected by COX. Because many anti-inflammatory and analgesic treatments, such as nonsteroidal anti-inflammatory drugs, rely on reduction in COX activity for their mechanism of action, the specific alterations observed in this study expand our understanding of the therapeutic and toxicologic consequences of COX inhibition.

COX-1和COX-2从蛛网膜化（AA）中产生前列腺素，并被认为在正常的脑功能中具有重要而独特的作用。 COX-1或COX-2的删除会导致前列腺素E2（PGE2）的大脑水平以及转录因子核因子-B（NF-KB）的激活都产生深远的差异，这表明COX-1和COX-C-2在脑蛛网膜酸代谢和基因表达的调节中都起着独特的作用。为了进一步阐明Cox同工型在脑转录组的调节中的作用，我们对缺乏COX-1（COX-1 KO）或COX-2（COX-2 KO）的小鼠的大脑皮层和海马中的基因表达进行了微阵列分析。尽管大多数基因（> 93％）显示在COX-1 KO或COX-2 KO小鼠的单独大脑区域中差异表达，仅在一种同工型的小鼠中出现在无效的小鼠中，而不是另一个同工型，但在Cox-1 KO和Cox-2 KO小鼠中，某些基因的表达都会改变。在脑皮质中，在COX-1 KO和COX-2 KO小鼠中有四个基因都有改变表达的基因。在这四种中，Rho-GDP解离抑制剂在两种基因型中均表现出增加。其他三个基因（线粒体内膜蛋白，GABA转运蛋白GAT3和RING FIRGER蛋白24）的表达在相反的方向上发生了变化（在COX-1 KO中下调，在COX-2 KO小鼠中上调，表明对这些基因表达的同工型特异性影响。 Such an isoform-specific effect was not observed in the hippocampus, where all 12 genes whose expression was altered in both COX-1 KO and COX-2 KO mice exhibited changes in a similar direction in both genotypes when compared with wild-type mice, suggesting that this cohort of genes is responsive to a general alteration in AA metabolism that is not specific to COX-1 or COX-2. In cerebral cortex of COX-2 KO mice, we identified increased expression of three genes that work in tandem in the final steps of short chain lipid metabolism by oxidation, namely hydroxyacyl-coenzyme A dehydrogenase type II (HADH2), acetyl-coenzyme A acetyltransferase 1 (ACAT1) and ATP citrate lyase (ACLY).在COX-1 KO小鼠的海马中发现ACAT 1的基因表达，但没有HADH2和ACLY的基因表达降低。在Cox-2 KO小鼠的大脑皮层中，蛋氨酸腺苷转移酶II（MAT2B）和腺基质类健康水解酶（AHCY）的表达增加了两个基因以将甲二氨氨酸代谢化为同型半胱氨酸。在COX-1 KO海马中，MAT2B的表达降低了，但不是Ahcy的表达。在Cox KO小鼠的脑皮质和海马中改变了两个参与GABA神经传递的基因的表达。 COX-2 KO小鼠海马中GABA-A受体亚基1（GABRB1）的微阵列分析表明基因表达的下调。用Q-PCR的验证表明，COX-2 KO小鼠海马中GABRB1的表达不仅降低，而且在COX-2 KO小鼠的脑皮质中也降低了。 Cox Ko小鼠脑皮质中的GABA转运蛋白（GAT）3表达表现出基因型特异性作用。 COX-1 KO小鼠表现出基因表达的下调，而Cox-2 KO显示mRNA水平的上调。发现Janus激酶（JAK）同工型1和2在海马中以基因型依赖性方式表达。 COX-1 KO小鼠的JAK1表达增加，但JAK2的表达不高。另一方面，COX-2 KO中的JAK-2表达降低，但在Cox-1 KO小鼠中却没有降低。总体而言，我们的发现表明，Cox活性的消融会改变许多基因的转录，包括参与氧化，蛋氨酸代谢，GABA神经传递和细胞因子信号传导的基因。尽管目前尚不清楚这些变化的一些分子机制，但这些数据识别了以前不受COX影响的代谢和信号传导途径。由于许多抗炎和镇痛治疗（例如非甾体类抗炎药）依赖于Cox活性的降低来降低COX的作用机制，因此在这项研究中观察到的特定变化扩展了我们对Cox抑制作用的治疗和毒性后果的理解。