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，炎症酶5-脂氧合酶(5-LOX)和环氧合酶-2(COX-2)在动脉粥样硬化、哮喘和许多类型的癌症中被诱导。这两种酶分别通过合成白三烯和前列腺素来调节这些疾病的炎症反应。白三烯和前列腺素途径传统上被视为独立的生物合成途径，因为通向这两个途径的关键步骤是共同底物花生四烯酸的初始氧化。这一建议是基于这一发现，即5-LOX产物5S-羟基二十碳四烯酸(5S-HETE)是COX-2(但不是COX-1同工酶)氧化的选择性和高效底物。COX-2这一新底物的发现将这两条二十烷类化合物途径紧密地联系在一起。常见的5-LOX/COX-2产物是一种结构类似于前列腺素H2但与前列腺素H2明显不同的新型双内源过氧化产物。在第一个特定目标中，我们建议通过建立反应动力学、底物专一性以及5-HETE与COX-2活性部位结合的结构基础来分析花生四烯酸转化为新型双内氧化物的酶学。将使用RAW264.7细胞和小鼠腹膜巨噬细胞研究双内过氧化产物的酶转化，RAW264.7细胞和小鼠腹膜巨噬细胞是同时表达5-LOX和COX-2的典型细胞。新二十烷类化合物及其代谢物家族的内源性形成将在小鼠动脉粥样硬化组织中进行评估。我们将检验这种新型双内过氧物(或其代谢物)具有抗炎特性的假设。初步研究表明，二内氧化物可抑制刺激的肥大细胞和微血管内皮细胞释放白介素8。将利用表达HMC-1的人肥大细胞5-LOX和COX-2来研究参与这种相互作用的信号通路。对新的5-LOX/COX-2交叉通路的表征无疑将使人们对5-LOX和COX-2的生物学有一个全新的认识，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线索。这些研究最终可能导致使用已有的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.