Sun D, Funk C D
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100, USA.
J Biol Chem. 1996 Sep 27;271(39):24055-62.
Previously, we isolated the murine "leukocyte-type" 12-lipoxygenase (L-12LO) cDNA from RNA of peritoneal-elicited cells that consisted predominantly of leukocytes (Chen, X.-S., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). By in situ hybridization we show that the L-12LO gene is expressed abundantly in a subset of peritoneal macrophages but not in elicited leukocytes, alveolar macrophages, or bone marrow-derived macrophages. L-12LO is highly related to human and rabbit 15-lipoxygenases, enzymes that have been implicated in the maturation process of red blood cells, and the oxidative modification of low density lipoproteins that is implicated in atherogenesis. Accordingly, these enzymes have been referred to as 12/15-lipoxygenases. We have inactivated the L-12LO gene in mice using homologous recombination in embryonic stem cells. Macrophage expression of L-12LO was abolished in homozygous deficient mice as was formation of 12-hydroxyeicosatetraenoic acid (12-HETE). In zymosan-stimulated cells, there was significant diversion of metabolism to the 5-lipoxygenase products leukotriene C4 and 5-HETE and in A23187-treated cells to 5-HETE only. The enhanced formation of 5-lipoxygenase metabolites was not due to compensatory changes of 5-lipoxygenase or 5-lipoxygenase activating protein but rather an apparent substrate diversion. L-12LO-deficient mice have no obvious abnormalities in reticulocyte or mature red blood cells, which suggest that in mice this pathway is not functionally important for erythrocytic development. Indices for oxidation of low density lipoprotein (measured as either thiobarbituric acid-reactive substances or the oxidant stress marker isoprostane 8-epi-prostaglandin F2alpha) were identical in incubations with unstimulated wild-type and L-12LO-deficient macrophages, but the zymosan-induced increase observed with wild-type macrophages was abolished in L-12LO-deficient cells. Thus, 12/15-lipoxygenase-deficient mice will be useful for the study of interaction between lipoxygenase pathways and determination of the in vivo role of 12/15-lipoxygenase-catalyzed oxidation of LDL in atherogenesis.
此前，我们从主要由白细胞组成的腹腔诱生细胞的RNA中分离出了小鼠“白细胞型”12 - 脂氧合酶（L - 12LO）cDNA（陈，X.-S.，库雷，U.，詹金斯，N. A.，科普兰，N. G.，和芬克，C. D.（1994年）《生物化学杂志》269，13979 - 13987）。通过原位杂交我们发现，L - 12LO基因在腹腔巨噬细胞的一个亚群中大量表达，但在诱生白细胞、肺泡巨噬细胞或骨髓来源的巨噬细胞中不表达。L - 12LO与人和兔的15 - 脂氧合酶高度相关，这些酶与红细胞的成熟过程以及与动脉粥样硬化发生有关的低密度脂蛋白的氧化修饰有关。因此，这些酶被称为12/15 - 脂氧合酶。我们利用胚胎干细胞中的同源重组使小鼠中的L - 12LO基因失活。在纯合缺陷小鼠中，L - 12LO的巨噬细胞表达被消除，12 - 羟基二十碳四烯酸（12 - HETE）的形成也被消除。在酵母聚糖刺激的细胞中，代谢显著转向5 - 脂氧合酶产物白三烯C4和5 - HETE，而在A23187处理的细胞中仅转向5 - HETE。5 - 脂氧合酶代谢产物形成的增加不是由于5 - 脂氧合酶或5 - 脂氧合酶激活蛋白的代偿性变化，而是明显的底物转向。L - 12LO缺陷小鼠的网织红细胞或成熟红细胞没有明显异常，这表明在小鼠中该途径对红细胞发育在功能上并不重要。低密度脂蛋白氧化指标（以硫代巴比妥酸反应性物质或氧化应激标志物异前列腺素8 - 表 - 前列腺素F2α测量）在未刺激的野生型和L - 12LO缺陷巨噬细胞的孵育中是相同的，但在L - 12LO缺陷细胞中，野生型巨噬细胞中观察到的酵母聚糖诱导的增加被消除。因此，12/15 - 脂氧合酶缺陷小鼠将有助于研究脂氧合酶途径之间的相互作用以及确定12/15 - 脂氧合酶催化的低密度脂蛋白氧化在动脉粥样硬化发生中的体内作用。
