Mechanisms for tissue specific expression and cell growth of 12/15-lipoxygenase

脂肪加氧酶（LOX）将分子氧结合到不饱和脂肪酸的特定碳原子中。根据花生四烯酸的氧合位点，哺乳动物组织中有5-、8-、12-和15- lox。根据其cdna的初级结构和酶学性质，它们主要分为5-LOX亚家族和12/15-LOX亚家族。5-LOX催化产生白三烯的第一步，白三烯在直接超敏反应和过敏中具有强大的生物活性。12/15-LOXs有多种亚型：白细胞、血小板和表皮。虽然12/15-LOXs已被证明在动脉粥样硬化和神经传递等多个系统中发挥作用，但其病理生理功能仍是研究和讨论的主题。有大量的间接证据表明LOX在低密度脂蛋白（LDL）的氧化修饰中起作用。本研究的目的是研究细胞内12-LOX在LDL氧化修饰导致泡沫细胞形成中的作用。我们在巨噬细胞样细胞系J774A.1中过表达12-脂氧合酶，该细胞系最初未显示可检测到的酶活性。将表达12-脂氧化酶的细胞与LDL在Dulbecco改良Eagle培养基中37℃孵育12 h后，硫代巴比妥酸反应物质测定LDL的氧化水平明显高于模拟转染细胞。用高效液相色谱法检测了改性LDL在碱性水解前后的氧化产物。大多数氧合衍生物为酯化形式，主要产物为13s -羟基十八烯酸- 9z， 11e -二烯酸。这些结果清楚地表明，在J774A.1细胞中表达的12-脂加氧酶可以氧化LDL中的酯化脂肪酸。此外，通过巨噬细胞降解实验评估，细胞内12-脂氧合酶产生的氧化LDL被清道夫受体识别。少

Lipoxygenase (LOX) incorporates a molecular oxygen into a specific carbon atom of unsaturated fatty acids. There are 5-, 8-, 12- and 15-LOXs in mammalian tissues according to the oxygenation site of arachidonic acid. Based upon the primary structures deduced from their cDNAs and enzymological properties, they are principally classified into 5-LOX and 12/15-LOX subfamilies. The 5-LOX catalyzes the first step in the generation of leukotrienes which have potent biological activities in the immediate hypersensitivity and allergy. There are number of isoforms of 12/15-LOXs : leukocyte, platelet and epidermis. Although the 12/15-LOXs have been shown to play roles in several systems such as atherosclerosis and neurotransmission, their pathophysiological functions are still subjects of investigation and discussion.There are a body of circumstantial evidences for a role of LOX in oxidative modification of low density lipoprotein (LDL). The aim of this study was to investigate the role of intrac … More ellular 12-LOX in the oxidative modification of LDL that leads to foam cell formation. We overexpressed 12-lipoxygenases in a macrophage-like cell line J774A.1 which originally did not show the detectable enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with LDL in Dulbecco's modified Eagle medium at 37 ℃ for 12 h, LDL oxidation as determined by thiobarbituric acid reactive substance was markedly increased over the mock-transfected cells. Oxygenated products in the modified LDL were examined by high performance liquid chromatography before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified to be 13S-hydroxyoctadeca-9Z, 11E-dienoic acid. These results clearly demonstrated that esterified fatty acids in LDL were oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay. Less