Convergence of the COX-2 and 5-lipoxygenase pathways

描述（由申请人提供）：有强有力的证据表明，在动脉粥样硬化、哮喘和许多类型的癌症中，炎症酶5-脂氧合酶（5-LOX）和环氧合酶-2 （COX-2）是诱导的。这两种酶分别通过合成白三烯和前列腺素介导这些疾病的炎症反应。白三烯和前列腺素途径传统上被认为是独立的生物合成途径，因为通往任何一条途径的承诺步骤都是共同底物花生四烯酸的初始氧化。这一提议是基于发现5-LOX产物5S-羟基二十碳四烯酸（5S- hete）是一种选择性和高效的底物，可被COX-2氧化（但不能被COX-1同工酶氧化）。这种新型COX-2底物的发现将这两种类二十烷途径紧密地联系在一起。常见的5-LOX/COX-2产物是一种新型的双内过氧化物，其结构与前列腺素H2相似，但明显不同。在第一个Specific Aim中，我们提出通过建立反应动力学、底物特异性和5-HETE在COX-2活性位点结合的结构基础，分析花生四烯酸转化为新型二内过氧化物的酶学。我们将使用RAW264.7细胞和小鼠腹膜巨噬细胞研究二内过氧化物产物的酶转化，这两种细胞都是表达5-LOX和COX-2的原型细胞。将在小鼠动脉粥样硬化组织中评估新型类二十烷酸及其代谢产物家族的内源性形成。我们将检验新的双内过氧化物（或其代谢物）具有抗炎特性的假设。初步研究表明，二内过氧化物可减弱受刺激肥大细胞和微血管内皮细胞释放的白细胞介素-8。参与这种相互作用的信号通路将使用表达HMC-1的5-LOX和COX-2的人肥大细胞进行研究。新的5-LOX/COX-2交叉通路的表征无疑将导致对5-LOX和COX-2生物学的全新理解，也将为动脉粥样硬化、哮喘和癌症等疾病的炎症成分的病因学和调控提供新的思路。这些研究可能最终导致使用现有抗炎药物治疗这些疾病的新治疗方案。

DESCRIPTION (provided by applicant): There is strong evidence for the induction of the inflammatory enzymes 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) in atherosclerosis, asthma, and many types of cancer. Both enzymes mediate the inflammatory response of these diseases through the synthesis of leukotrienes and prostaglandins, respectively. The leukotriene and prostaglandin pathways have traditionally been viewed as independent biosynthetic routes since the committed step toward either pathway is taken as the initial oxygenation of the common substrate, arachidonic acid. This proposal is based on the finding that the 5-LOX product, 5S- hydroxyeicosatetraenoic acid (5S-HETE), is a selective and highly efficient substrate for oxygenation by COX-2 (but not by the COX-1 isozyme). The discovery of this novel substrate for COX-2 intimately links the two eicosanoid pathways. The common 5-LOX/COX-2 product is a novel di-endoperoxide that is structurally reminiscent of, but distinctly different from prostaglandin H2. In the first Specific Aim we propose to analyze the enzymology of the conversion of arachidonic acid into the novel di-endoperoxide by establishing the reaction kinetics, substrate specificity, and the structural basis for binding of 5-HETE in the COX-2 active site. The enzymatic transformation of the di-endoperoxide product will be studied using RAW264.7 cells and murine peritoneal macrophages, prototypical cells that expresses both 5-LOX and COX-2. Endogenous formation of the novel eicosanoid and its family of metabolites will be assessed in mouse atherosclerotic tissue. We will test the hypothesis that the novel di-endoperoxide (or its metabolites) possess anti- inflammatory properties. Preliminary studies have shown that the di-endoperoxide attenuates the release of interleukin-8 from stimulated mast cells and microvascular endothelial cells. The signaling pathways involved in this interaction will be studied using the 5-LOX and COX-2 expressing HMC-1 human mast cell. Characterization of the novel 5-LOX/COX-2 cross-over pathway will undoubtedly lead to an entirely new understanding of the biology of 5-LOX and COX-2, and it will also shed new light on the etiology and regulation of the inflammatory component of diseases like atherosclerosis, asthma, and cancer. These studies could ultimately lead to new therapeutic regimens for the treatment of these diseases using established anti-inflammatory medications.